Monoclonal antibodies and their use

ABSTRACT

Isolated monoclonal antibodies are disclosed herein that specifically bind a cell surface antigen expressed on the human pancreatic endocrine cells or a subset thereof, and/or a precursor thereof. Isolated monoclonal antibodies are also disclosed herein that specifically bind a cell surface antigen expressed on human pancreatic exocrine cells or human ductal cells. Humanized forms of these antibodies, and functional fragments of these antibodies, are also disclosed. The antibodies can be conjugated to an effector molecule, or a detectable marker. These antibodies are of use to detect and/or isolate pancreatic cells or a subset thereof. Methods of treating a pancreatic tumor are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.13/010,737, filed Jan. 20, 2011, which is a divisional of U.S. patentapplication Ser. No. 12/298,761, filed Oct. 27, 2008, now issued as U.S.Pat. No. 7,981,628, which is the U.S. National Stage of PCT ApplicationNo. PCT/US2007/010466, filed Apr. 27, 2007, which was published inEnglish under PCT Article 21(2), which in turn claims the benefit ofU.S. Provisional Application No. 60/795,900, filed Apr. 28, 2006. Theprior applications are incorporated herein by reference in theirentirety.

STATEMENT OF GOVERNMENT SUPPORT

This invention was made with United States government support, pursuantto grant 1U01DK072477 from the National Institutes of Health (NIDDK).The United States government has certain rights in the invention.

FIELD

This application relates to the fields of diabetes and cancer,specifically to antibodies that specifically bind an antigen expressedon the surface of human pancreatic endocrine cells, exocrine cells, andductal cells.

BACKGROUND

A mammalian pancreas is composed of two subclasses of tissue: theexocrine cells of the acinar tissue and the endocrine cells of theislets of Langerhans. The exocrine cells produce digestive enzymes thatare secreted through the pancreatic duct to the intestine. The isletcells produce polypeptide hormones that are involved in carbohydratemetabolism. The islands of endocrine tissue that exist within the adultmammalian pancreas are termed the islets of Langerhans. Adult mammalianislets are composed of four major cell types, the α, β, δ, and PP cells.These cells are distinguished by their production of glucagon, insulin,somatostatin, and pancreatic polypeptide, respectively.

Diabetes mellitus results from the failure of cells to transportendogenous glucose across their membranes either because of anendogenous deficiency of insulin or an insulin receptor defect. Diabetestype 1, or insulin dependent diabetes mellitus (IDDM) is caused by thedestruction of β cells, which results in insufficient levels ofendogenous insulin. Diabetes type 2, or non-insulin dependent diabetes,is believed to be a defect in either the insulin receptor itself or inthe number of insulin receptors present or in the balance betweeninsulin and glucagon signals. Although diabetes runs in families, and avariety of heritable mutations have been implicated in the developmentof the disease, no single genetic marker has been identified that isresponsible for this condition.

Current treatment of individuals with clinical manifestation of diabetesattempts to emulate the role of the pancreatic β cells in a non-diabeticindividual. Individuals with normal β cell function exhibit preciseregulation of insulin secretion in response to serum glucose levels.This regulation is due to a feedback mechanism that resides in the βcells that ordinarily prevents surges of blood sugar outside of thenormal limits. Unless blood sugar is controlled properly, dangerous oreven fatal levels can result. Hence, treatment of a diabetic individualinvolves monitoring of blood glucose levels and the use of injectedbovine, porcine, or cloned human insulin as required. Despite suchintervention, there is often a gradual decline in the health ofdiabetics.

Diabetes afflicts millions of people in the United States alone, andthere is a clear need to provide cells capable of replacing pancreaticendocrine function. The ability to isolate distinct populations of livepancreatic endocrine cells represents a key step towards achieving thisgoal. This permits in vitro modeling of the Islet of Langerhans, for thestudy of normal and aberrant glucose metabolism and facilitate theisolation and/or evaluation of β cells. In addition, there is a need toproduce new clinical treatments for diabetes, including the productionof islet cells for transplantation (see U.S. Pat. No. 4,439,521; U.S.Pat. No. 5,510,263; U.S. Pat. No. 5,646,035; U.S. Pat. No. 5,961,972).Successful transplants of whole isolated islets, for example, have beenmade in animals and in humans. The success of the Edmonton protocol inthe treatment of type 1 diabetes highlighted the promise of cellularreplacement therapy for this disorder (Hirshberg et al., Rev EndocrMetab Disord. 4:381-389, 2003; Sharpiro et al., N Engl J. Med.343:230-238, 2000). Unfortunately, insulin independence has notnecessarily been shown to be durable in transplant recipients.

There is a need to identify and isolate islet cells or islet progenitorcells that can be used for β cell expansion or differentiation in vitroor for direct transplantation. Furthermore, there is a need fordiagnostic methods that can accurately assess the number of pancreaticendocrine cells (or a subset thereof) in a subject, such as a subjectwith type 1 or type 2 diabetes.

SUMMARY

Isolated monoclonal antibodies are disclosed herein that specificallybind a cell surface antigen expressed on distinct subsets of humanpancreatic cells. In one embodiment, an antibody is provided thatspecifically bind endocrine cells or a subset thereof, and/or aprecursor thereof. The antibody does not bind a pancreatic endocrinehormone, such as insulin, glucagon, somatostatin or pancreaticpolypeptide, or their receptors, and do not bind pancreatic exocrinetissue. In another embodiment, a monoclonal antibody is provided thatspecifically binds a cell surface antigen on pancreatic duct cells.These antibodies do not bind endocrine or exocrine cells. In a thirdembodiment, a monoclonal antibody is disclosed that specifically binds acell surface antigen on exocrine cells. The antibody does not bindendocrine or duct cells.

Humanized forms of these antibodies, and functional fragments of theseantibodies, are also disclosed. The antibodies can be conjugated to aneffector molecule, such as a detectable marker, a therapeutic agent, ora toxin.

In one example, the isolated monoclonal antibodies bindinsulin-producing cell (β cells). In another example, the isolatedmonoclonal antibodies bind glucagon-producing cells (α cells). Inadditional examples, the isolated monoclonal antibodies bind a cellsurface antigen that is expressed on the cell surface of one or more ofinsulin producing cells, somatostatin-producing cells, pancreaticpolypeptide producing cells and glucagon producing cells. In a furtherexample, the isolated monoclonal antibodies bind all types of pancreaticendocrine cells. These antibodies are of use to detect and isolate allpancreatic endocrine cells, or subsets of pancreatic endocrine cells. Inseveral examples, the isolated monoclonal antibodies can be used todetect diabetes or a pancreatic endocrine cell tumor. In an additionalembodiment, the isolated monoclonal antibodies bind a cell surfaceantigen on pancreatic ductal cells, and can be used to detect pancreaticadenocarcinoma. In a further embodiment, the islolated monoclonalantibodies bind a cell surface antigen on pancreatic exocrine cells, andcan be used to detect a pancreatic exocrine tumor. The antibodies can beused for in vitro or in vivo detection of pancreatic cells or a subsetthereof.

Methods of treating a pancreatic endocrine tumor, a pancreatic exocrinetumor, or a pancreatic adenocarcinoma are also disclosed herein. Themethod includes administering a therapeutically effective amount of oneor more of these antibodies to a subject with the pancreatic tumor.

In additional embodiments, antibodies that specifically bind duct andexocrine can be used to isolate of purified populations of these cellsto initiate cell cultures that can be modified to express insulin.Furthermore, methods for isolating purified populations of endocrinecells are disclosed, wherein positive selection for endocrine cells iscombined with negative selection for markers of duct and exocrine cells.

The foregoing and other features and advantages will become moreapparent from the following detailed description of several embodiments,which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a set of digital images obtained using monoclonal antibodiesdirected against antigens associated with pancreatic cells. Each panelin this figure illustrates staining with antibody from an independentlyderived hybridoma. Panels HIC0 4-F9, HIC1 2-B4, HIC1 4-G6, HIC1 5-F10and HIC1 7-H10 illustrate staining of all islet cells (pan-islet). Thepanel labeled HIC0 3-05 illustrates staining with an antibody that bindsto a subset of endocrine cells (including a subset of (3 cells). Panelslabeled DHIC2 2-B4 and DHIC2 2-C12 illustrate staining with antibodiesthat react with alpha cells, although DHIC2 2-B4 likely reacts to alesser extent with other pancreatic endocrine cells. Antibodies withduct-specific labeling are illustrated in panels DHIC2 4-A10 and DHIC35-H10, and exocrine acinar labeling is shown in panel HIC1 1-C10.Sections of pancreas were incubated with primary antibodies, then with aCy-3-conjugated anti-mouse Ig (H+L) antibody. Cells reacting withprimary antibody appear light against the dark background of cells thatdo not react with the antibody.

FIG. 2 is a set of plots showing that antibodies directed againstpancreatic cells react with cell surface molecules. Flow cytometricanalyses of viable (non-fixed) enzyme-dispersed pancreatic cellsrevealed that all of the antibody-producing hybridomas react with cellsurface molecules. Enzyme-dispersed pancreatic cells were incubated withpancreatic cell specific antibodies, and antibody reactivity wasassessed by flow cytometry using an APC-conjugated F(ab′)₂ fragmentanti-mouse immunoglobulin (H+L).

FIGS. 3A-3D are diagrams showing the production of humanized antibodies.FIG. 3A is a schematic diagram of an antibody showing the disulfidelinked heavy (H) and light (L) chains, and the location of regionswithin the ˜110 amino acid variable (V) domains that show a high degreeof sequence variation. These regions determine the antigen specificityand are called “hypervariable regions” or “complementarity determiningregions,” (CDRs). FIG. 3B is a bar graph illustrating the variability ofthe three hypervariable regions CDR1-CDR3, with sequence variabilityplotted as a function of residue number along the polypeptide chain. Thethree large peaks correspond to the three hypervariable regions in (A).FIG. 3C is the amino acid sequence of the HIC0 4-F9 monoclonal antibodyV_(H) (SEQ ID NO: 39) aligned with the humanized D3h44 Fab (SEQ ID NO:40, PDB accession#1JPT; 1.85 Å resolution). The intervening sequence isthe amino acids that are identical in HIC0 4-F9 and D3h44 (SEQ ID NOs:39 and 40). FIG. 3D is a ribbon diagram of the D3h44 Fv_(H). Thestructure is based on the crystallographic coordinates of the D3h44 Fab(PDB accession code 1JPT). The amino and carboxyl termini of the D3h44Fv_(H) are labeled N, C, respectively. The conserved cysteines thatcomprise the disulfide bond are rendered as space-filled models, and thehypervariable regions CDR1-CDR3 are indicated as loops 1, 2, and 3, withthe framework regions also indicated

FIG. 4 is a plot of a FACS analysis that illustrates the separation ofcells from an islet culture by FACS on the basis of their level ofbinding by HIC1 7-H10. FIG. 4A shows the non-specific cell fluorescenceassociated with random binding of the secondary antibody alone. FIGS.4B-D show the binding of HIC1 7-H10 in the pre-sort population,negatively sorted population and positively sorted populationrespectively. Sorting and analysis of these cells was performed using aCytopeia Influx™. Detection of the primary antibody labeling wasachieved with an APC-conjugated F(ab′)₂ fragment of goat anti-mouseimmunoglobulin (H+L).

FIG. 5 is a set of digital images obtained using monoclonal antibodiesthat specifically bind antigens associated with pancreatic duct cellsand pancreatic adenocarcinoma cells. The monoclonal antibodies DHIC24-A10 and DHIC3 5-H10 were incubated with sections of normal pancreas(left panels) and pancreatic adenocarcinoma (right panels), and aperoxidase-conjugated polyclonal secondary antibody was used to detectsection-bound unlabeled primary antibody. Addition of the chromogen3,3′-diaminobenzidine tetrahydrochloride (DAB) allowed visualization ofbound antibody (dark gray to black) as well as areas where with noantibody binding (light areas). Stained sections were analyzed by lightmicroscopy.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand.

The Sequence Listing is submitted as an ASCII text file[899-71968-08_Sequence_Listing.txt, Dec. 13, 2011, 20.9 KB], which isincorporated by reference herein

DETAILED DESCRIPTION I. Abbreviations

-   -   CDR: complementarity determining region    -   dsFv: disulfide stabilized fragment of a variable region    -   DMEM: Dulbecco's modified eagle medium    -   ELISA: enzyme-linked immunosorbent assay    -   EM: effector molecule    -   Fab′: antigen binding immunoglobulin fragment    -   F(ab)′₂: divalent antigen binding immunoglobulin fragment    -   FACS: fluorescence activated cell sorting    -   Fv: fragment of a variable region    -   kDa: kilodaltons    -   LCDR: light chain complementarity determining region    -   HCDR: heavy chain complementarity determining region    -   Ig: immunoglobulin    -   MAb: monoclonal antibody    -   PBS: phosphate buffered saline    -   PP: pancreatic polypeptide    -   scFv: single chain fragment of a variable region    -   SDR: specificity determining residues    -   SDS-PAGE: sodium dodecyl (lauryl) sulfate-polyacrylamide gel        electrophoresis    -   RIA: radioimmunoas says    -   V_(H): variable region of a heavy chain    -   V_(L): variable region of a light chain

II. Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments of thisdisclosure, the following explanations of specific terms are provided:

α cells: Mature glucagon producing cells. In vivo, these cells are foundin the pancreatic islets of Langerhans.

β cells: Mature insulin producing cells. In vivo, these cells are foundin the pancreatic islets of Langerhans,

δ cells: Mature somatostatin producing cells. In vivo, these cells arefound in the pancreatic islets of Langerhans.

PP cells: Mature pancreatic polypeptide (PP) producing cells. In vivo,these cells are found in the pancreatic islets of Langerhans.

Amplification: Of a nucleic acid molecule (such as, a DNA or RNAmolecule) refers to use of a technique that increases the number ofcopies of a nucleic acid molecule in a specimen. An example ofamplification is the polymerase chain reaction, in which a biologicalsample collected from a subject is contacted with a pair ofoligonucleotide primers, under conditions that allow for thehybridization of the primers to a nucleic acid template in the sample.The primers are extended under suitable conditions, dissociated from thetemplate, and then re-annealed, extended, and dissociated to amplify thenumber of copies of the nucleic acid. The product of amplification maybe characterized by electrophoresis, restriction endonuclease cleavagepatterns, oligonucleotide hybridization or ligation, and/or nucleic acidsequencing using standard techniques. Other examples of amplificationinclude strand displacement amplification, as disclosed in U.S. Pat. No.5,744,311; transcription-free isothermal amplification, as disclosed inU.S. Pat. No. 6,033,881; repair chain reaction amplification, asdisclosed in WO 90/01069; ligase chain reaction amplification, asdisclosed in EP-A-320 308; gap filling ligase chain reactionamplification, as disclosed in U.S. Pat. No. 5,427,930; and NASBA™ RNAtranscription-free amplification, as disclosed in U.S. Pat. No.6,025,134.

Animal: Living multi-cellular vertebrate organisms, a category thatincludes, for example, mammals and birds. The term mammal includes bothhuman and non-human mammals. Similarly, the term “subject” includes bothhuman and veterinary subjects.

Antibody: A polypeptide ligand comprising at least a light chain orheavy chain immunoglobulin variable region which specifically recognizesand binds an epitope of an antigen or a fragment thereof. Antibodies arecomposed of a heavy and a light chain, each of which has a variableregion, termed the variable heavy (V_(H)) region and the variable light(V_(L)) region. Together, the V_(H) region and the V_(L) region areresponsible for binding the antigen recognized by the antibody.

This includes intact immunoglobulins and the variants and portions ofthem well known in the art, such as Fab′ fragments, F(ab)′₂ fragments,single chain Fv proteins (“scFv”), and disulfide stabilized Fv proteins(“dsFv”). A scFv protein is a fusion protein in which a light chainvariable region of an immunoglobulin and a heavy chain variable regionof an immunoglobulin are bound by a linker, while in dsFvs, the chainshave been mutated to introduce a disulfide bond to stabilize theassociation of the chains. The term also includes genetically engineeredforms such as chimeric antibodies (for example, humanized murineantibodies), heteroconjugate antibodies (such as, bispecificantibodies). See also, Pierce Catalog and Handbook, 1994-1995 (PierceChemical Co., Rockford, Ill.); Kuby, J., Immunology, 3^(rd) Ed., W.H.Freeman & Co., New York, 1997.

Typically, a naturally occurring immunoglobulin has heavy (H) chains andlight (L) chains interconnected by disulfide bonds. There are two typesof light chain, lambda (λ) and kappa (k). There are five main heavychain classes (or isotypes) which determine the functional activity ofan antibody molecule: IgM, IgD, IgG, IgA and IgE.

Each heavy and light chain contains a constant region and a variableregion, (the regions are also known as “domains”). In combination, theheavy and the light chain variable regions specifically bind theantigen. Light and heavy chain variable regions contain a “framework”region interrupted by three hypervariable regions, also called“complementarity-determining regions” or “CDRs”. The extent of theframework region and CDRs have been defined (see, Kabat et al.,Sequences of Proteins of Immunological Interest, U.S. Department ofHealth and Human Services, 1991, which is hereby incorporated byreference). The Kabat database is now maintained online. The sequencesof the framework regions of different light or heavy chains arerelatively conserved within a species. The framework region of anantibody, that is the combined framework regions of the constituentlight and heavy chains, serves to position and align the CDRs inthree-dimensional space. The CDRs are primarily responsible for bindingto an epitope of an antigen.

The CDRs of each chain are typically referred to as CDR1, CDR2, andCDR3, numbered sequentially starting from the N-terminus, and are alsotypically identified by the chain in which the particular CDR islocated. Thus, a V_(H) CDR3 is located in the variable domain of theheavy chain of the antibody in which it is found, whereas a V_(L) CDR1is the CDR1 from the variable domain of the light chain of the antibodyin which it is found. An antibody that binds an antigen of interest hasa specific V_(H) region and the V_(L) region sequence, and thus specificCDR sequences. Antibodies with different specificities (due to differentcombining sites for different antigens) have different CDRs. Although itis the CDRs that vary from antibody to antibody, only a limited numberof amino acid positions within the CDRs are directly involved in antigenbinding. These positions within the CDRs are called specificitydetermining residues (SDRs).

References to “V_(H)” or “VH” refer to the variable region of animmunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.References to “V_(L)” or “VL” refer to the variable region of animmunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.

A “monoclonal antibody” is an antibody produced by a single clone ofB-lymphocytes or by a cell into which the light and heavy chain genes ofa single antibody have been transfected, or a progeny thereof.Monoclonal antibodies are produced by methods known to those of skill inthe art, for instance by making hybrid antibody-forming cells from afusion of myeloma cells with immune spleen cells. Monoclonal antibodiesinclude humanized monoclonal antibodies.

A “chimeric antibody” has framework residues from one species, such ashuman, and CDRs (which generally confer antigen binding) from anotherspecies, such as a murine antibody that specifically binds a cellsurface antigen on a pancreatic endocrine cell.

A “humanized” immunoglobulin is an immunoglobulin including a humanframework region and one or more CDRs from a non-human (for example amouse, rat, or synthetic) immunoglobulin. The non-human immunoglobulinproviding the CDRs is termed a “donor,” and the human immunoglobulinproviding the framework is termed an “acceptor.” In one embodiment, allthe CDRs are from the donor immunoglobulin in a humanizedimmunoglobulin. Constant regions need not be present, but if they are,they must be substantially identical to human immunoglobulin constantregions, such as at least about 85-90%, such as about 95% or moreidentical. Hence, all parts of a humanized immunoglobulin, exceptpossibly the CDRs, are substantially identical to corresponding parts ofnatural human immunoglobulin sequences. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin. A humanized antibody binds to the same antigen as thedonor antibody that provides the CDRs. The acceptor framework of ahumanized immunoglobulin or antibody may have a limited number ofsubstitutions by amino acids taken from the donor framework. Humanizedor other monoclonal antibodies can have additional conservative aminoacid substitutions which have substantially no effect on antigen bindingor other immunoglobulin functions. Humanized immunoglobulins can beconstructed by means of genetic engineering (see for example, U.S. Pat.No. 5,585,089).

Artificial Islets: Clusters of pancreatic endocrine cells formed by thedifferentiation of stem or progenitor cells including ES cell in vitro,dislodged clusters of pancreatic endocrine cells, endocrine cellsdifferentiated from stem cells or progenitor cells including ES cells invitro, cells that have undergone a mesenchymal-to-epithelial orepithelial-to-mesenchymal-to-epithelial transition or endocrine cellsaggregated into a cluster in vitro.

Binding affinity: Affinity of an antibody for an antigen. In oneembodiment, affinity is calculated by a modification of the Scatchardmethod described by Frankel et al., Mol. Immunol., 16:101-106, 1979. Inanother embodiment, binding affinity is measured by an antigen/antibodydissociation rate. In yet another embodiment, a high binding affinity ismeasured by a competition radioimmunoas say. In several examples, a highbinding affinity is at least about 1×10⁻⁸ M. In other embodiments, ahigh binding affinity is at least about 1.5×10⁻⁸ M, at least about2.0×10⁻⁸M, at least about 2.5×10⁻⁸M, at least about 3.0×10⁻⁸M, at leastabout 3.5×10⁻⁸M, at least about 4.0×10⁻⁸M, at least about 4.5×10⁻⁸M, orat least about 5.0×10⁻⁸ M.

Chimeric antibody: An antibody which includes sequences derived from twodifferent antibodies, which typically are of different species. Mosttypically, chimeric antibodies include human and murine antibodydomains, generally human constant regions and murine variable regions,murine CDRs and/or murine SDRs.

Complementarity Determining Region (CDR): Amino acid sequences whichtogether define the binding affinity and specificity of the natural Fvregion of a native Ig binding site. The light and heavy chains of an Igeach have three CDRs, designated L-CDR1, L-CDR2, L-CDR3 and H-CDR1,H-CDR2, H-CDR3, respectively. By definition, the CDRs of the light chainare bounded by the residues at positions 24 and 34 (L-CDR1), 50 and 56(L-CDR2), 89 and 97 (L-CDR3); the CDRs of the heavy chain are bounded bythe residues at positions 31 and 35b (H-CDR1), 50 and 65 (H-CDR2), 95and 102 (H-CDR3), using the numbering convention delineated by Kabat etal., (1991) Sequences of Proteins of Immunological Interest, 5^(th)Edition, U.S. Department of Health and Human Services, Public HealthService, National Institutes of Health, Bethesda, Md. (NIH PublicationNo. 91-3242).

Contacting: Placement in direct physical association. Includes both insolid and liquid form.

Differentiation: The process whereby relatively unspecialized cells(e.g., embryonic cells) acquire specialized structural and/or functionalfeatures characteristic of mature cells. Similarly, “differentiate”refers to this process. Typically, during differentiation, cellularstructure alters and tissue-specific proteins appear. The term“differentiated pancreatic endocrine cell” refers to cells expressing aprotein characteristic of the specific pancreatic endocrine cell type. Adifferentiated pancreatic endocrine cell includes an α cell, a β cell, aδ cell, and a PP cell, which express glucagon, insulin, somatostatin,and pancreatic polypeptide, respectively.

Growth factor: A substance that promotes cell growth, survival, and/ordifferentiation. Growth factors include molecules that function asgrowth stimulators (mitogens), molecules that function as growthinhibitors (e.g. negative growth factors) factors that stimulate cellmigration, factors that function as chemotactic agents or inhibit cellmigration or invasion of tumor cells, factors that modulatedifferentiated functions of cells, factors involved in apoptosis, orfactors that promote survival of cells without influencing growth anddifferentiation. Examples of growth factors are fibroblast growth factor(FGF)₂, epidermal growth factor (EGF), ciliary neurotrophic factor(CNTF), hepatocyte growth factor (HGF), nerve growth factor (NGF), andactvin-A.

Effective amount or Therapeutically effective amount: The amount ofagent or cells that is an amount sufficient to prevent, treat (includingprophylaxis), reduce and/or ameliorate the symptoms and/or underlyingcauses of any of a disorder or disease. In one embodiment, an “effectiveamount” is sufficient to reduce or eliminate a symptom of a disease,such as a pancreatic cancer. In another embodiment, an effective amountis an amount sufficient to overcome the disease itself, such as indiabetes.

Effector molecule: The portion of a chimeric molecule that is intendedto have a desired effect on a cell to which the chimeric molecule istargeted. Effector molecule is also known as an effector moiety (EM),therapeutic agent, or diagnostic agent, or similar terms.

Therapeutic agents include such compounds as nucleic acids, proteins,peptides, amino acids or derivatives, glycoproteins, radioisotopes,lipids, carbohydrates, or recombinant viruses. Nucleic acid therapeuticand diagnostic moieties include antisense nucleic acids, derivatizedoligonucleotides for covalent cross-linking with single or duplex DNA,and triplex forming oligonucleotides. Alternatively, the molecule linkedto a targeting moiety, such as an antibody, may be an encapsulationsystem, such as a liposome or micelle that contains a therapeuticcomposition such as a drug, a nucleic acid (such as an antisense nucleicacid), or another therapeutic moiety that can be shielded from directexposure to the circulatory system. Means of preparing liposomesattached to antibodies are well known to those of skill in the art. See,for example, U.S. Pat. No. 4,957,735; and Connor et al., Pharm. Ther.28:341-365, 1985. Diagnostic agents or moieties include radioisotopesand other detectable labels. Detectable labels useful for such purposesare also well known in the art, and include radioactive isotopes such as³²P, ¹²⁵I, and ¹³¹I, fluorophores, chemiluminescent agents, magneticresonance imaging agents and enzymes.

Endocrine: Tissue which secretes regulatory hormones directly into thebloodstream without the need for an associated duct system.

Epitope: An antigenic determinant. These are particular chemical groupsor peptide sequences on a molecule that are antigenic, i.e. that elicita specific immune response. An antibody specifically binds a particularantigenic epitope on a polypeptide.

Epithelial-to-Mesenchymal Transition: The epithelium is the covering ofinternal and external surfaces of the body, including the lining ofvessels and other small cavities that consists of cells joined bybiological cementing substances. Generally, fully differentiatedepithelial cells express proteins characteristic of a differentiatedphenotype, such as insulin, and have a limited capacity to proliferate.The mesenchyme is the meshwork of loosely organized embryonic connectivetissue in the mesoderm from which are formed the connective tissues ofthe body, along with the blood vessels and lymphatic vessels. An“epithelial-to-mesenchymal” transition is a biological process in whichcells of an epithelial origin, such as pancreatic endocrine cells, adoptthe characteristics of mesenchymal cells. A “mesenchymal-to-epithelial”transition is the reverse process, wherein mesencymal cells adoptcharacteristics of epithelial cells. An“epithelial-to-mesenchymal-to-epithelial” transition can occur whenepithelial cells transiently adopt mesenchymal characteristics beforeresuming epithelial characteristics. These processes can be induced incell culture.

Expand: A process by which the number or amount of cells in a cellculture is increased due to cell division. Similarly, the terms“expansion” or “expanded” refers to this process. The terms“proliferate,” “proliferation” or “proliferated” may be usedinterchangeably with the words “expand,” “expansion”, or “expanded.”Typically, during an expansion phase, the cells do not differentiate toform mature cells.

Expressed: Translation of a nucleic acid into a protein. Proteins may beexpressed and remain intracellular, become a component of the cellsurface membrane, or be secreted into the extracellular matrix ormedium.

Exocrine: Secretory tissue which distributes its products, such asenzymes, via an associated duct network. The exocrine pancreas is thepart of the pancreas that secretes enzymes required for digestion. Theexocrine cells of the pancreas include the centroacinar cells andbasophilic cells, which produce secretin and cholecystokinin.

Expression Control Sequences: Nucleic acid sequences that regulate theexpression of a heterologous nucleic acid sequence to which it isoperatively linked. Expression control sequences are operatively linkedto a nucleic acid sequence when the expression control sequences controland regulate the transcription and, as appropriate, translation of thenucleic acid sequence. Thus expression control sequences can includeappropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons. The term “controlsequences” is intended to include, at a minimum, components whosepresence can influence expression, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences. Expression control sequences can include apromoter.

A promoter is a minimal sequence sufficient to direct transcription.Also included are those promoter elements which are sufficient to renderpromoter-dependent gene expression controllable for cell-type specific,tissue-specific, or inducible by external signals or agents; suchelements may be located in the 5′ or 3′ regions of the gene. Bothconstitutive and inducible promoters are included (see for example,Bitter et al., Methods in Enzymology 153:516-544, 1987). For example,when cloning in bacterial systems, inducible promoters such as pL ofbacteriophage lambda, plac, ptrp, ptac (ptrp-lac hybrid promoter) andthe like may be used. In one embodiment, when cloning in mammalian cellsystems, promoters derived from the genome of mammalian cells (such asmetallothionein promoter) or from mammalian viruses (such as theretrovirus long terminal repeat; the adenovirus late promoter; thevaccinia virus 7.5K promoter) can be used. Promoters produced byrecombinant DNA or synthetic techniques may also be used to provide fortranscription of the nucleic acid sequences.

Framework Region: Amino acid sequences interposed between CDRs. Includesvariable light and variable heavy framework regions. The frameworkregions serve to hold the CDRs in an appropriate orientation for antigenbinding.

Heterologous: A heterologous sequence is a sequence that is not normally(in the wild-type sequence) found adjacent to a second sequence. In oneembodiment, the sequence is from a different genetic source, such as avirus or organism, than the second sequence.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Immunogenic peptide: A peptide which comprises an allele-specific motifor other sequence, such as an N-terminal repeat, such that the peptidewill bind an MHC molecule and induce a cytotoxic T lymphocyte (“CTL”)response, or a B cell response (such as antibody production) against theantigen from which the immunogenic peptide is derived.

In one embodiment, immunogenic peptides are identified using sequencemotifs or other methods, such as neural net or polynomialdeterminations, known in the art. Typically, algorithms are used todetermine the “binding threshold” of peptides to select those withscores that give them a high probability of binding at a certainaffinity and will be immunogenic. The algorithms are based either on theeffects on MHC binding of a particular amino acid at a particularposition, the effects on antibody binding of a particular amino acid ata particular position, or the effects on binding of a particularsubstitution in a motif-containing peptide. Within the context of animmunogenic peptide, a “conserved residue” is one which appears in asignificantly higher frequency than would be expected by randomdistribution at a particular position in a peptide. In one embodiment, aconserved residue is one where the MHC structure may provide a contactpoint with the immunogenic peptide.

Immunologically reactive conditions: Includes reference to conditionswhich allow an antibody raised against a particular epitope to bind tothat epitope (or cell expressing the epitope) to a detectably greaterdegree than, and/or to the substantial exclusion of, binding tosubstantially all other epitopes (or cells not expressing the epitope).Immunologically reactive conditions are dependent upon the format of theantibody binding reaction and typically are those utilized inimmunoassay protocols or those conditions encountered in vivo. SeeHarlow & Lane, supra, for a description of immunoassay formats andconditions. The immunologically reactive conditions employed in themethods are “physiological conditions” which include reference toconditions (such as temperature, osmolarity, pH) that are typical insidea living mammal or a mammalian cell. While it is recognized that someorgans are subject to extreme conditions, the intra-organismal andintracellular environment normally lies around pH 7 (i.e., from pH 6.0to pH 8.0, more typically pH 6.5 to 7.5), contains water as thepredominant solvent, and exists at a temperature above 0° C. and below50° C. Osmolarity is within the range that is supportive of cellviability and proliferation.

Islets of Langerhans: Small discrete clusters of pancreatic endocrinetissue. In vivo, in an adult mammal, the islets of Langerhans are foundin the pancreas as discrete clusters (islands) of pancreatic endocrinetissue surrounded by the pancreatic exocrine (or acinar) tissue. Invivo, the islets of Langerhans consist of the α cells, β cells, δ cells,and PP cells. Histologically, the islets of Langerhans consist of acentral core of β cells surrounded by an outer layer of a cells, δcells, and PP cells. The islets of Langerhans are sometimes referred toherein as “islets.”

Isolated: An “isolated” biological component (such as a nucleic acid,peptide or protein) has been substantially separated, produced apartfrom, or purified away from other biological components in the cell ofthe organism in which the component naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA, and proteins. Nucleicacids, peptides and proteins which have been “isolated” thus includenucleic acids and proteins purified by standard purification methods.The term also embraces nucleic acids, peptides and proteins prepared byrecombinant expression in a host cell as well as chemically synthesizednucleic acids. An isolated cell type has been substantially separatedfrom other cell types, such as a different cell type that occurs in anorgan. A purified cell or component can be at least 90%, at least 95%,at least 96%, at least 97%, at least 98%, or at least 99% pure.

Label: A detectable compound or composition that is conjugated directlyor indirectly to another molecule, such as an antibody or a protein, tofacilitate detection of that molecule. Specific, non-limiting examplesof labels include fluorescent tags, enzymatic linkages, and radioactiveisotopes.

Linker peptide: A peptide within an antibody binding fragment (such asan Fv fragment) which serves to indirectly bond the variable heavy chainto the variable light chain. “Linker” can also refer to a peptideserving to link a targeting moiety, such as a scFv, to an effectormolecule, such as a cytotoxin or a detectable label.

The terms “conjugating,” “joining,” “bonding” or “linking” refer tomaking two polypeptides into one contiguous polypeptide molecule, or tocovalently attaching a radionuclide or other molecule to a polypeptide,such as an scFv. In the specific context, the terms include reference tojoining a ligand, such as an antibody moiety, to an effector molecule(“EM”). The linkage can be either by chemical or recombinant means.“Chemical means” refers to a reaction between the antibody moiety andthe effector molecule such that there is a covalent bond formed betweenthe two molecules to form one molecule.

Lymphocytes: A type of white blood cell that is involved in the immunedefenses of the body. There are two main types of lymphocytes: B cellsand T cells.

Mammal: This term includes both human and non-human mammals. Similarly,the term “subject” includes both human and veterinary subjects.

Nucleic acid: A polymer composed of nucleotide units (ribonucleotides,deoxyribonucleotides, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof) linked viaphosphodiester bonds, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof. Thus, the termincludes nucleotide polymers in which the nucleotides and the linkagesbetween them include non-naturally occurring synthetic analogs, such as,for example and without limitation, phosphorothioates, phosphoramidates,methyl phosphonates, chiral-methyl phosphonates, 2-O-methylribonucleotides, peptide-nucleic acids (PNAs), and the like. Suchpolynucleotides can be synthesized, for example, using an automated DNAsynthesizer. The term “oligonucleotide” typically refers to shortpolynucleotides, generally no greater than about 50 nucleotides. It willbe understood that when a nucleotide sequence is represented by a DNAsequence (i.e., A, T, G, C), this also includes an RNA sequence (i.e.,A, U, G, C) in which “U” replaces “T.”

Conventional notation is used herein to describe nucleotide sequences:the left-hand end of a single-stranded nucleotide sequence is the5′-end; the left-hand direction of a double-stranded nucleotide sequenceis referred to as the 5′-direction. The direction of 5′ to 3′ additionof nucleotides to nascent RNA transcripts is referred to as thetranscription direction. The DNA strand having the same sequence as anmRNA is referred to as the “coding strand;” sequences on the DNA strandhaving the same sequence as an mRNA transcribed from that DNA and whichare located 5′ to the 5′-end of the RNA transcript are referred to as“upstream sequences;” sequences on the DNA strand having the samesequence as the RNA and which are 3′ to the 3′ end of the coding RNAtranscript are referred to as “downstream sequences.”

“cDNA” refers to a DNA that is complementary or identical to an mRNA, ineither single stranded or double stranded form.

“Encoding” refers to the inherent property of specific sequences ofnucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA, toserve as templates for synthesis of other polymers and macromolecules inbiological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA produced by that geneproduces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and non-codingstrand, used as the template for transcription, of a gene or cDNA can bereferred to as encoding the protein or other product of that gene orcDNA. Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

“Recombinant nucleic acid” refers to a nucleic acid having nucleotidesequences that are not naturally joined together. This includes nucleicacid vectors comprising an amplified or assembled nucleic acid which canbe used to transform a suitable host cell. A host cell that comprisesthe recombinant nucleic acid is referred to as a “recombinant hostcell.” The gene is then expressed in the recombinant host cell toproduce, such as a “recombinant polypeptide.” A recombinant nucleic acidmay serve a non-coding function (such as a promoter, origin ofreplication, ribosome-binding site, etc.) as well.

A first sequence is an “antisense” with respect to a second sequence ifa polynucleotide whose sequence is the first sequence specificallyhybridizes with a polynucleotide whose sequence is the second sequence.

Terms used to describe sequence relationships between two or morenucleotide sequences or amino acid sequences include “referencesequence,” “selected from,” “comparison window,” “identical,”“percentage of sequence identity,” “substantially identical,”“complementary,” and “substantially complementary.”

For sequence comparison of nucleic acid sequences, typically onesequence acts as a reference sequence, to which test sequences arecompared. When using a sequence comparison algorithm, test and referencesequences are entered into a computer, subsequence coordinates aredesignated, if necessary, and sequence algorithm program parameters aredesignated. Default program parameters are used. Methods of alignment ofsequences for comparison are well known in the art. Optimal alignment ofsequences for comparison can be conducted, for example, by the localhomology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, bythe homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.48:443, 1970, by the search for similarity method of Pearson & Lipman,Proc. Nat'l. Acad. Sci. USA 85:2444, 1988, by computerizedimplementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA inthe Wisconsin Genetics Software Package, Genetics Computer Group, 575Science Dr., Madison, Wis.), or by manual alignment and visualinspection (see for example, Current Protocols in Molecular Biology(Ausubel et al., eds 1995 supplement)).

One example of a useful algorithm is PILEUP. PILEUP uses asimplification of the progressive alignment method of Feng & Doolittle,J. Mol. Evol. 35:351-360, 1987. The method used is similar to the methoddescribed by Higgins & Sharp, CABIOS 5:151-153, 1989. Using PILEUP, areference sequence is compared to other test sequences to determine thepercent sequence identity relationship using the following parameters:default gap weight (3.00), default gap length weight (0.10), andweighted end gaps. PILEUP can be obtained from the GCG sequence analysissoftware package, such as version 7.0 (Devereaux et al., Nuc. Acids Res.12:387-395, 1984.

Another example of algorithms that are suitable for determining percentsequence identity and sequence similarity are the BLAST and the BLAST2.0 algorithm, which are described in Altschul et al., J. Mol. Biol.215:403-410, 1990 and Altschul et al., Nucleic Acids Res. 25:3389-3402,1977. Software for performing BLAST analyses is publicly availablethrough the National Center for Biotechnology Information(http://www.ncbi.nlm.nih.gov/). The BLASTN program (for nucleotidesequences) uses as defaults a word length (W) of 11, alignments (B) of50, expectation (E) of 10, M=5, N=−4, and a comparison of both strands.The BLASTP program (for amino acid sequences) uses as defaults a wordlength (W) of 3, and expectation (E) of 10, and the BLOSUM62 scoringmatrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89:10915,1989).

Operably linked: A first nucleic acid sequence is operably linked with asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked DNA sequences are contiguousand, where necessary to join two protein coding regions, in the samereading frame.

ORF (open reading frame): A series of nucleotide triplets (codons)coding for amino acids without any termination codons. These sequencesare usually translatable into a peptide.

Pancreatic endocrine cell: An endocrine cell of pancreatic origin thatproduces one or more pancreatic hormone, such as insulin, glucagon,somatostatin, or pancreatic polypeptide. Subsets of pancreatic endocrinecells include the α (glucagons producing), β (insulin producing) δ(somatostatin producing) or PP (pancreatic polypeptide producing) cells.Additional subsets produce more than one pancreatic hormone, such as,but not limited to, a cell that produces both insulin and glucagon, or acell that produces insulin, glucagon, and somatostatin, or a cell thatproduces insulin and somatostatin.

Pancreatic cancer: A malignant tumor within the pancreas. The prognosisis generally poor. About 95% of pancreatic cancers are adenocarcinomas.The remaining 5% are tumors of the exocrine pancreas (for example,serous cystadenomas), ascinar cell cancers, and pancreaticneuroendocrine tumors (such as insulinomas). An “insulinoma” is a cancerof the beta cells that retains the ability to secrete insulin. Patientswith insulinomas usually develop neuroglycopenic symptoms. These includerecurrent headache, lethargy, diplopia, and blurred vision, particularlywith exercise or fasting. Severe hypoglycemia may result in seizures,coma and permanent neurological damage. Symptoms resulting from thecatecholaminergic response to hypoglycemia (for example, tremulousness,palpitations, tachycardia, sweating, hunger, anxiety, nausea). Apancreatic adenocarciona occurs in the glandular tissue. Symptomsinclude abdominal pain, loss of appetite, weight loss, jaundice andpainless extension of the gallbladder.

Classical treatment for pancreatic cancer, including adenocarcinomas andinsulinomas includes surgical resection (such as the Whipple procedure)and chemotherapy with agent such as fluorouracil, gemcitabine, anderlotinib.

Polypeptide: A polymer in which the monomers are amino acid residuesthat are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The terms “polypeptide” or“protein” as used herein is intended to encompass any amino acidsequence and include modified sequences such as glycoproteins. The term“polypeptide” is specifically intended to cover naturally occurringproteins, as well as those that are recombinantly or syntheticallyproduced.

The term “polypeptide fragment” refers to a portion of a polypeptidewhich exhibits at least one useful epitope. The term “functionalfragments of a polypeptide” refers to all fragments of a polypeptidethat retain an activity of the polypeptide. Biologically functionalfragments, for example, can vary in size from a polypeptide fragment assmall as an epitope capable of binding an antibody molecule to a largepolypeptide capable of participating in the characteristic induction orprogramming of phenotypic changes within a cell. An “epitope” is aregion of a polypeptide capable of binding an immunoglobulin generatedin response to contact with an antigen. Thus, smaller peptidescontaining the biological activity of insulin, or conservative variantsof the insulin, are thus included as being of use.

The term “soluble” refers to a form of a polypeptide that is notinserted into a cell membrane.

The term “substantially purified polypeptide” as used herein refers to apolypeptide which is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Conservative substitutions replace one amino acid with another aminoacid that is similar in size, hydrophobicity, etc. Variations in thecDNA sequence that result in amino acid changes, whether conservative ornot, should be minimized in order to preserve the functional andimmunologic identity of the encoded protein. The immunologic identity ofthe protein may be assessed by determining if it is recognized by anantibody; a variant that is recognized by such an antibody isimmunologically conserved. Any cDNA sequence variant will preferablyintroduce no more than twenty, and preferably fewer than ten amino acidsubstitutions into the encoded polypeptide. Variant amino acid sequencesmay, for example, be 80, 90 or even 95% or 98% identical to the nativeamino acid sequence.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers useful in this invention are conventional. Remington'sPharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton,Pa., 15th Edition (1975), describes compositions and formulationssuitable for pharmaceutical delivery of the fusion proteins hereindisclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example sodiumacetate or sorbitan monolaurate.

Pharmaceutical agent: A chemical compound or a composition capable ofinducing a desired therapeutic or prophylactic effect when properlyadministered to a subject or a cell. “Incubating” includes a sufficientamount of time for a drug to interact with a cell. “Contacting” includesincubating a drug in solid or in liquid form with a cell.

Polynucleotide: A nucleic acid sequence (such as a linear sequence) ofany length. Therefore, a polynucleotide includes oligonucleotides, andalso gene sequences found in chromosomes. An “oligonucleotide” is aplurality of joined nucleotides joined by native phosphodiester bonds.An oligonucleotide is a polynucleotide of between 6 and 300 nucleotidesin length. An oligonucleotide analog refers to moieties that functionsimilarly to oligonucleotides but have non-naturally occurring portions.For example, oligonucleotide analogs can contain non-naturally occurringportions, such as altered sugar moieties or inter-sugar linkages, suchas a phosphorothioate oligodeoxynucleotide. Functional analogs ofnaturally occurring polynucleotides can bind to RNA or DNA, and includepeptide nucleic acid (PNA) molecules.

Primers: Short nucleic acids, for example DNA oligonucleotides 10nucleotides or more in length, which are annealed to a complementarytarget DNA strand by nucleic acid hybridization to form a hybrid betweenthe primer and the target DNA strand, then extended along the target DNAstrand by a DNA polymerase enzyme. Primer pairs can be used foramplification of a nucleic acid sequence, such as by the polymerasechain reaction (PCR) or other nucleic-acid amplification methods knownin the art.

Probes and primers as used in the present invention may, for example,include at least 10 nucleotides of the nucleic acid sequences that areshown to encode specific proteins. In order to enhance specificity,longer probes and primers may also be employed, such as probes andprimers that comprise 15, 20, 30, 40, 50, 60, 70, 80, 90 or 100consecutive nucleotides of the disclosed nucleic acid sequences. Methodsfor preparing and using probes and primers are described in thereferences, for example Sambrook et al. (1989) Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, N.Y.; Ausubel et al. (1987)Current Protocols in Molecular Biology, Greene Publ. Assoc. &Wiley-Intersciences; Innis et al. (1990) PCR Protocols, A Guide toMethods and Applications, Innis et al. (Eds.), Academic Press, SanDiego, Calif. PCR primer pairs can be derived from a known sequence, forexample, by using computer programs intended for that purpose such asPrimer (Version 0.5, 1991, Whitehead Institute for Biomedical Research,Cambridge, Mass.).

When referring to a probe or primer, the term specific for (a targetsequence) indicates that the probe or primer hybridizes under stringentconditions substantially only to the target sequence in a given samplecomprising the target sequence.

Promoter: A promoter is an array of nucleic acid control sequences whichdirect transcription of a nucleic acid. A promoter includes necessarynucleic acid sequences near the start site of transcription, such as, inthe case of a polymerase II type promoter, a TATA element. A promoteralso optionally includes distal enhancer or repressor elements which canbe located as much as several thousand base pairs from the start site oftranscription.

Recombinant: A recombinant nucleic acid is one that has a sequence thatis not naturally occurring or has a sequence that is made by anartificial combination of two otherwise separated segments of sequence.This artificial combination is often accomplished by chemical synthesisor, more commonly, by the artificial manipulation of isolated segmentsof nucleic acids, such as by genetic engineering techniques. Similarly,a recombinant protein is one encoded for by a recombinant nucleic acidmolecule.

Selectively hybridize: Hybridization under moderately or highlystringent conditions that excludes non-related nucleotide sequences.

In nucleic acid hybridization reactions, the conditions used to achievea particular level of stringency will vary, depending on the nature ofthe nucleic acids being hybridized. For example, the length, degree ofcomplementarity, nucleotide sequence composition (such as GC versus ATcontent), and nucleic acid type (such as RNA versus DNA) of thehybridizing regions of the nucleic acids can be considered in selectinghybridization conditions. An additional consideration is whether one ofthe nucleic acids is immobilized, for example, on a filter.

A specific, non-limiting example of progressively higher stringencyconditions is as follows: 2×SSC/0.1% SDS at about room temperature(hybridization conditions); 0.2×SSC/0.1% SDS at about room temperature(low stringency conditions); 0.2×SSC/0.1% SDS at about 42° C. (moderatestringency conditions); and 0.1×SSC at about 68° C. (high stringencyconditions). One of skill in the art can readily determine variations onthese conditions (see Molecular Cloning: A Laboratory Manual, 2nd ed.,Vol. 1-3, ed. Sambrook et al., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., 1989). Washing can be carried out using only one ofthese conditions, for example, high stringency conditions, or each ofthe conditions can be used, for example, for 10-15 minutes each, in theorder listed above, repeating any or all of the steps listed. However,as mentioned above, optimal conditions will vary, depending on theparticular hybridization reaction involved, and can be determinedempirically.

Sequence identity of amino acid sequences: The similarity between aminoacid sequences is expressed in terms of the similarity between thesequences, otherwise referred to as sequence identity. Sequence identityis frequently measured in terms of percentage identity (or similarity orhomology); the higher the percentage, the more similar the two sequencesare. Homologs or variants of a polypeptide will possess a relativelyhigh degree of sequence identity when aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smithand Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J.Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci.U.S.A. 85:2444, 1988; Higgins and Sharp, Gene 73:237, 1988; Higgins andSharp, CABIOS 5:151, 1989; Corpet et al., Nucleic Acids Research16:10881, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85:2444, 1988. Altschul et al., Nature Genet. 6:119, 1994, presents adetailed consideration of sequence alignment methods and homologycalculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403, 1990) is available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe NCBI website on the internet.

Homologs and variants of a V_(L) or a V_(H) of an antibody are typicallycharacterized by possession of at least about 75%, for example at leastabout 80%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity counted overthe full length alignment with the amino acid sequence of the antibodyusing the NCBI Blast 2.0, gapped blastp set to default parameters. Forcomparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). When aligning short peptides (fewerthan around 30 amino acids), the alignment should be performed using theBlast 2 sequences function, employing the PAM30 matrix set to defaultparameters (open gap 9, extension gap 1 penalties). Proteins with evengreater similarity to the reference sequences will show increasingpercentage identities when assessed by this method, such as at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least99% sequence identity. When less than the entire sequence is beingcompared for sequence identity, homologs and variants will typicallypossess at least 80% sequence identity over short windows of 10-20 aminoacids, and may possess sequence identities of at least 85% or at least90% or 95% depending on their similarity to the reference sequence.Methods for determining sequence identity over such short windows areavailable at the NCBI website on the internet. One of skill in the artwill appreciate that these sequence identity ranges are provided forguidance only; it is entirely possible that strongly significanthomologs could be obtained that fall outside of the ranges provided.

Specific binding agent: An agent that binds substantially only to adefined target. Thus a β cell specific binding agent is an agent thatbinds substantially to a β cell, and a pancreatic endocrine cellspecific binding agent is an gent that binds substantially only topancreatic endocrine cells or a subset thereof (and not to pancreaticexocrine cells). Similarly, a pancreatic exocrine cell specific bindingagent is an agent that binds substantially to exocrine cells. In oneembodiment, the specific binding agent is a monoclonal or polyclonalantibody that specifically binds a type of pancreatic cell.

The term “specifically binds” refers, with respect to a cell, such as apancreatic endocrine cell, to the preferential association of anantibody or other ligand, in whole or part, with a cell or tissuebearing that antigen and not to cells or tissues lacking that antigen.It is, of course, recognized that a certain degree of non-specificinteraction may occur between a molecule and a non-target cell ortissue. Nevertheless, specific binding may be distinguished as mediatedthrough specific recognition of the antigen. Although selectivelyreactive antibodies bind antigen, they may do so with low affinity. Onthe other hand, specific binding results in a much stronger associationbetween the antibody (or other ligand) and cells bearing the antigenthan between the bound antibody (or other ligand) and cells lacking theantigen. Specific binding typically results in greater than 2-fold, suchas greater than 5-fold, greater than 10-fold, or greater than 100-foldincrease in amount of bound antibody or other ligand (per unit time) toa cell or tissue expressing the target epitope as compared to a cell ortissue lacking this epitope. Specific binding to a protein under suchconditions requires an antibody that is selected for its specificity fora particular protein. A variety of immunoassay formats are appropriatefor selecting antibodies or other ligands specifically immunoreactivewith a particular protein. For example, solid-phase ELISA immunoassaysare routinely used to select monoclonal antibodies specificallyimmunoreactive with a protein. See Harlow & Lane, Antibodies, ALaboratory Manual, Cold Spring Harbor Publications, New York (1988), fora description of immunoassay formats and conditions that can be used todetermine specific immunoreactivity.

Stem cell: A cell that can generate fully differentiated functionalcells of more than one given cell type. The role of stem cells in vivois to replace cells that are destroyed during the normal life of ananimal. Generally, stem cells are pluripotent and can divide withoutlimit. After division, the stem cell may remain as a stem cell, become aprecursor cell, or proceed to terminal differentiation. Althoughappearing morphologically unspecialized, the stem cell may be considereddifferentiated where the possibilities for further differentiation arelimited. An embryonic stem cell is one type of stem cell. A“tissue-specific stem cell” (an adult stem cell) is another type of stemcell, one that gives rise to cells of a specific tissue type. Aprecursor cell is a cell that can generate a fully differentiatedfunctional cell of at least one given cell type. Precursor cells candivide, but have a limited ability to give rise to new precursor cells.A “pancreatic precursor cell” is a precursor cell that gives rise to oneor more cell types within the pancreas. In one embodiment, a pancreaticendocrine precursor cell gives rise to all of the pancreatic endocrinecells (α cells, β cells, δ cells, and PP cells), but does not give riseto other cells, such as the pancreatic exocrine cells. In anotherembodiment, a pancreatic precursor cell gives rise to more than one typeof pancreatic endocrine cell, although it may not give rise to all ofthe types of endocrine cells. One specific, non-limiting example of apancreatic precursor cell is a β precursor cell that give rise to bothdifferentiated α and β cells.

Subject: Any mammal, such as humans, non-human primates, pigs, sheep,cows, rodents and the like which is to be the recipient of theparticular treatment. In two non-limiting examples, a subject is a humansubject or a murine subject.

T Cell: A white blood cell critical to the immune response. T cellsinclude, but are not limited to, CD4⁺ T cells and CD8⁺ T cells. A CD4⁺ Tlymphocyte is an immune cell that carries a marker on its surface knownas “cluster of differentiation 4” (CD4). These cells, also known ashelper T cells, help orchestrate the immune response, including antibodyresponses as well as killer T cell responses. CD8⁺ T cells carry the“cluster of differentiation 8” (CD8) marker. In one embodiment, CD8 Tcells are cytotoxic T lymphocytes. In another embodiment, a CD8 cell isa suppressor T cell.

Therapeutic agent: Used in a generic sense, it includes treating agents,prophylactic agents, and replacement agents. A therapeutic agent can bean antibody that specifically binds pancreatic endocrine cells or asubset thereof.

Transduced and Transformed: A virus or vector “transduces” a cell whenit transfers nucleic acid into the cell. A cell is “transformed” or“transfected” by a nucleic acid transduced into the cell when the DNAbecomes stably replicated by the cell, either by incorporation of thenucleic acid into the cellular genome, or by episomal replication.

Numerous methods of transfection are known to those skilled in the art,such as: chemical methods (e.g., calcium-phosphate transfection),physical methods (e.g., electroporation, microinjection, particlebombardment), fusion (e.g., liposomes), receptor-mediated endocytosis(e.g., DNA-protein complexes, viral envelope/capsid-DNA complexes) andby biological infection by viruses such as recombinant viruses {Wolff,J. A., ed, Gene Therapeutics, Birkhauser, Boston, USA (1994)}. In thecase of infection by retroviruses, the infecting retrovirus particlesare absorbed by the target cells, resulting in reverse transcription ofthe retroviral RNA genome and integration of the resulting provirus intothe cellular DNA. Methods for the introduction of genes into thepancreatic endocrine cells are known (e.g. see U.S. Pat. No. 6,110,743,herein incorporated by reference). These methods can be used totransduce a pancreatic endocrine cell produced by the methods describedherein, or an artificial islet produced by the methods described herein.

Genetic modification of the target cell is an indicium of successfultransfection. “Genetically modified cells” refers to cells whosegenotypes have been altered as a result of cellular uptakes of exogenousnucleotide sequence by transfection. A reference to a transfected cellor a genetically modified cell includes both the particular cell intowhich a vector or polynucleotide is introduced and progeny of that cell.

Transgene: An exogenous gene supplied by a vector.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in the host cell, such as anorigin of replication. A vector may also include one or more therapeuticgenes and/or selectable marker genes and other genetic elements known inthe art. A vector can transduce, transform or infect a cell, therebycausing the cell to express nucleic acids and/or proteins other thanthose native to the cell. A vector optionally includes materials to aidin achieving entry of the nucleic acid into the cell, such as a viralparticle, liposome, protein coating or the like.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this disclosure belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. It is further tobe understood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of this disclosure, suitable methods andmaterials are described below. The term “comprises” means “includes.”All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Monoclonal Antibodies that Bind Pancreatic Cell Surface Antigens

Isolated monoclonal antibodies are disclosed herein that specificallybind cell surface antigens on a human pancreatic endocrine cell, a humanpancreatic endocrine cell precursor, or both. The antibody can bind alltypes of pancreatic endocrine cells, or a subset thereof. In oneexample, the monoclonal antibody binds antigens on the cell surface ofpancreatic endocrine cells (or a subset thereof) of an adult human. Inanother example, the monoclonal antibody binds a cell surface antigenexpressed on an islet of Langerhans, either from an adult human or froma fetus. In one embodiment, the antibodies do not bind a pancreaticendocrine hormone, such as insulin, glucagon, somatostatin or pancreaticpolypeptide, or their receptors. In a further embodiment, the monoclonalantibodies that specifically bind pancreatic endocrine cells (or asubset thereof) do not bind pancreatic exocrine cells and/or pancreaticductal cells. In some examples, the antibodies specifically bind humanpancreatic endocrine cells. In additional examples the antibodiesspecifically bind human pancreatic endocrine cells and specifically bindpancreatic endocrine cells of a non-human primate. These antibodies donot bind pancreatic exocrine cells.

In one example, the isolated monoclonal antibodies bindinsulin-producing cell (β cells) or a subset of these cells. Inadditional examples, the isolated monoclonal antibodies bind a cellsurface antigen that is expressed on the cell surface of one or more ofinsulin producing cells, somatostatin producing cells, pancreaticpolypeptide producing cells and glucagon producing cells. In furtherexamples, the monoclonal antibody binds glucagon producing cells, orglucagon producing cells and insulin producing cells. In an additionalexample, the antibody binds all types of pancreatic endocrine cells(insulin, glucagon, somatostatin, and pancreatic polypeptide producingpancreatic endocrine cells).

Also disclosed herein are isolated monoclonal antibodies that bind cellsurface antigens that are specific to pancreatic exocrine cells orpancreatic ductal cells. In one example, the monoclonal antibodyspecifically binds antigens on the cell surface of pancreatic exocrinecells (or a subset thereof) of an adult human and/or a fetus. In anotherexample, the monoclonal antibody specifically binds a cell surfaceantigen expressed on a pancreatic ductal cell, either from an adulthuman or from a fetus. In one embodiment, the antibodies thatspecifically bind a pancreatic ductal cell or a pancreatic exocrine celldo not specifically bind pancreatic endocrine cells, such as cells thatproduce insulin, glucagon, somatostatin or pancreatic polypeptide. Insome examples, the antibodies specifically bind human pancreaticexocrine cells, and do not bind pancreatic endocrine cells or pancreaticductal cells. In additional examples, the antibodies specifically bindhuman pancreatic ductal cells, but do not bind pancreatic exocrine cellsor pancreatic ductal cells.

Generally, the monoclonal antibodies each include a variable heavy(V_(H)) and a variable light (V_(L)) chain and specifically bind thecell surface antigen. For example, the antibody can specifically bindpancreatic endocrine cells, and can bind the cell surface antigen ofendocrine cells with an affinity constant of at least 10⁶ M⁻¹, such asat least 10⁷ M⁻¹, at least 10⁸ M⁻¹, at least 5×10⁸ M⁻¹, or at least 10⁹M⁻¹. In a second example, the antibody can specifically bind pancreaticexocrine cells, and can bind the cell surface antigen of exocrine cellswith an affinity constant of at least 10⁶ M⁻¹, such as at least 10⁷ M⁻¹,at least 10⁸ M⁻¹, at least 5×10⁸ M⁻¹, or at least 10⁹ M⁻¹. In a thirdexample, the antibody can specifically bind pancreatic ductal cells, andcan bind the cell surface antigen of ductal cells with an affinityconstant of at least 10⁶ M⁻¹, such as at least 10⁷ M⁻¹, at least 10⁸M⁻¹, at least 5×10⁸ M⁻¹, or at least 10⁹ M⁻¹.

It should be noted that antibody fragments are encompassed by thepresent disclosure. Thus the isolated monoclonal antibody can be, forexample, a Fab′ fragment, a F(ab)′₂ fragment, a single chain Fv protein(“scFv”), or a disulfide stabilized Fv protein (“dsFv”).

Monoclonal antibodies that specifically bind pancreatic endocrine cellsor a subset thereof can bind the antigen recognized by an antibodyproduced by one or more of hybridomas HIC0 4-F9, HIC1 2-B4, HIC1 4-G6,HIC1 7-H10, HIC0 3-05, HIC1 5-F10, DHIC2 2-B4, or DHIC2 2-C12. Patentdeposits of hybridomas HIC0 3-05, HIC0 4-F9, DHIC2 2-B4 and DHIC 2-C12,HIC1 5-F10, HIC1 7-H10, HIC1-2-B4 and HIC1 4-G6 were made with theAmerican Type Culture Collection (ATCC), 10801 University Blvd.Manassas, Va. 20110-2209 on Apr. 27, 2006, in accordance with theBudapest Treaty (Accession Nos. PTA-7556, PTA-7557, PTA-7558, PTA-7559,PTA-7560, PTA-7561, PTA-7562 and PTA-7563, respectively). It should benoted that a “monoclonal antibody produced by one or more hybridomas”refers to antibodies produced by the hybridoma or any progeny thereof.

In several embodiments, the heavy chain of the variable region of themonoclonal antibody includes one or more of the following CDR sequences.

TABLE 1 CDR 1 CDR 2 CDR 3 HIC0 DYYIH WIDPENGNTIYDPKFQD YYGSTYYFDY 4-F9(SEQ ID NO: (SEQ ID NO: 2) (SEQ ID NO: 3) 1) HIC0 EYI IHWFYPGSGGLKYSEKFK HEKYFDY 5-F10 (SEQ ID NO: D (SEQ ID NO: 6) 4)(SEQ ID NO: 5) HIC0 NYAMS TISSGGSYTYYPDSVKG QGDNYWYFDV 3-C5 (SEQ ID NO:(SEQ ID NO: 8) (SEQ ID NO: 9) 7) HIC1 DYYMH FIRNKANGYTTEYSASV DIKGDY4-G6 (SEQ ID NO: G (SEQ ID NO: 12) 10) (SEQ ID NO: 11) HIC1 SYDMSYISSGGGSTYFPNTVKG HGGNQPWFAY 7-H10 (SEQ ID NO: (SEQ ID NO: 14)(SEQ ID NO: 15) 13) DHIC2 TDYSMH NTETGEPTYADDFKG GYGSSSWFAY 2-C12(SED ID NO: (SEQ ID NO: 36) (SEQ ID NO: 37) 35)

In additional embodiment, the antibody includes a heavy chain comprisingthe amino acid sequences listed in Table 2, or an amino acid sequence atleast 95%, 96%, 97%, 98% or 99% identical to one of these sequences.

TABLE 2* Hybridoma ID Full heavy chain V-region sequence HIC0 4-F9EVQLQQSGAELVRPGALVKLSCKASGFNIKDYYIHWVKQKPEQGLEWIGWIDPENGNTIYDPKFQDKASITSDTSSNTAYLQLSSLTSEDTAVYYCTSYYGSTYYFDYWGQGT (SEQ ID NO: 39) HIC0 3-C5EVMLVESGGGLVKPGGSLKLSCAASGFTFSNYAMSWVRQTPEKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCARQGDNYWYFDVWGAGTTVTVSSESQ (SEQ ID NO: 16) HIC1 5-F10VQLQQSGAELVKPGASVKLSCKASGYTFTEYIIHWVKQRSGQGLEWIGWFYPGSGGLKYSEKFKDKATLTADKSSSTVYMELSRLTSEDSAVYFCARHEKYFDYWGQGTT (SEQ ID NO: 17) HIC1 4-G6EVKLVESGGGLVQPGGSLRLSCATSGFTFTDYYMHWVRQPPGKALEWLGFIRNKANGYTTEYSASVKGRFTISRDNSQSILYLQMNTLRAEDSATYYCTRDIKGDYWGQGTS (SEQ ID NO: 18) HIC1 7-H10EVQLVESGGGLVKPGGSLKLSCAASGFAFSSYDMSWVRQAPDKRLEWVAYISSGGGSTYFPNTVKGRFTISRDNAKNTLSLQMSSLRSEDTAMYYCTRHGGNQPWFAYWGQGT (SEQ ID NO: 19) DHIC2 2-QVQIQLVQSGPELKKPGETVKISCKASGYTFTDYSMHWVKQAPGKG C12LKWMGWINTETGEPTYADDFKGRFAFSLETSASTAYLQINNLKNEDTATYFCSRGYGSSSWFAYWGQGTLVT (SEQ ID NO: 38) *CDR amino acid sequences areunderlined.

In several embodiments, the antibody includes a heavy chain comprisingone of the CDRs listed in Table 1, or a combination of these CDRs. Onespecific example is an antibody including a heavy chain comprising SEQID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3 or a combination thereof. Inadditional examples the antibody includes a heavy chain comprising SEQID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or a combination thereof. Infurther examples, the antibody includes a heavy chain comprising SEQ IDNO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or a combination thereof. In yetanother example, the antibody includes a heavy chain comprising SEQ IDNO: 10, SEQ ID NO: 11, SEQ ID NO: 12, or a combination thereof. Inanother example, the antibody includes a heavy chain comprising SEQ IDNO: 13, SEQ ID NO: 14, SEQ ID NO: 15 or a combination thereof. Theantibody can include a heavy chain comprising at least one of SEQ IDNOs: 1-15, wherein the antibody specifically binds pancreatic endocrinecells, or a subset thereof.

In several embodiments the antibody includes a V_(H) polypeptideincluding amino acid sequences of CDRs that are at least about 90%identical, such as at least about 95%, at least about 98%, or at leastabout 99% identical to the amino acid sequence set forth as SEQ ID NO:1, SEQ ID NO: 2 and SEQ ID NO: 3, or amino acid sequences of CDRs thatare at least about 90% identical, such as at least about 95%, at leastabout 98%, or at least about 99% identical to the amino acid sequenceset forth as SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, or a V_(H)polypeptide having amino acid sequences of the CDRs that are at leastabout 90% identical, such as at least about 95%, at least about 98%, orat least about 99% identical to the amino acid sequence set forth as SEQID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. In additional embodiments theantibody includes a V_(H) polypeptide including amino acid sequences ofCDRs that are at least about 90% identical, such as at least about 95%,at least about 98%, or at least about 99% identical to the amino acidsequence set forth as SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, oramino acid sequences of CDRs that are at least about 90% identical, suchas at least about 95%, at least about 98%, or at least about 99%identical to the amino acid sequence set forth as SEQ ID NO: 13, SEQ IDNO: 14 and SEQ ID NO: 15. In a further embodiment, the antibody includesV_(H) polypeptide including an amino acid sequence set forth as one ofSEQ ID NOs: 16-18 and 38-39 (see also FIG. 3).

In additional embodiment, the antibody includes the CDR sequences frommonoclonal antibodies that specifically bind the pancreatic endocrinecell antigen recognized by an antibody secreted by the hybridoma HIC04-F9, HIC1 2-B4, HIC1-4-G6, HIC1 7-H10, HIC0 3-05, HIC1 5-F10, DHIC22-B4, or DHIC2 2-C12. Patent deposits of hybridomas HIC0 3-05, HIC04-F9, DHIC2 2-B4 and DHIC 2-C12, HIC1 5-F10, HIC1 7-H10, HIC1 2-B4 andHIC1 4-G6 were made with the American Type Culture Collection (ATCC)10801 University Blvd., Manassas, Va. 20110-2209 on Apr. 27, 2006, inaccordance with the Budapest Treaty (Accession Nos. PTA-7556, PTA-7557,PTA-7558, PTA-7559, PTA-7560, PTA-7561, PTA-7562 and PTA-7563,respectively). The framework region included in the antibody that bindspancreatic endocrine cells (or a subset thereof) can be the endogenousframework region (from monoclonal antibodies produced by HIC0 4-F9, HIC12-B4, HIC1 4-G6, HIC1 7-H10, HIC0 3-05, HIC1 5-F10, DHIC2 2-B4, or DHIC22-C12, respectively). Alternatively, a heterologous framework region,such as, but not limited to a human framework region, can be included inthe heavy and/or light chain of the antibodies.

In yet another embodiment the monoclonal antibody specifically bindsductal cells, such as an antibody secreted by DHIC2 4-A10 or DHIC35-H10. These hybridomas were deposited with the ATCC in accordance withthe Budapest Treaty on Apr. 26, 2007. It should be noted that a“monoclonal antibody produced by one or more of hybridomas DHIC2 4-A10or DHIC3 5-H10” refers to antibodies produced by these hybridomas or anyprogeny thereof.

Thus, the antibody that specifically binds pancreatic ductal cells caninclude the CDR sequences from monoclonal antibodies that specificallybind the ductal cell surface antigen secreted by the hybridoma DHIC24-A10 or DHIC3 5-H10. The framework region included in the antibody thatbinds pancreatic endocrine cells (or a subset thereof) can be theendogenous framework region (from monoclonal antibodies produced byDHIC2 4-A10 or DHIC3 5-H10 respectively). Alternatively, a heterologousframework region, such as, but not limited to a human framework region,can be included in the heavy and/or light chain of the antibodies.

In a further embodiment the monoclonal antibody specifically bindsexocrine cells, such as the antibody secreted by HIC1 1-C10. Thishybridoma was deposited with ATCC in accordance with the Budapest Treatyon Apr. 26, 2007. It should be noted that a “monoclonal antibodyproduced by the hybridom HIC1 1-C10” refers to antibodies produced bythese hybridomas or any progeny thereof.

Thus, the antibody that specifically binds pancreatic exocrine cells caninclude the CDR sequences from monocolanal antibodies that specificallybind the exocrine cells surface antigen secreted by the hybridoma HIC11-C10. The framework region included in the antibody that bindspancreatic endocrine cells (or a subset thereof) can be the endogenousframework region (from monoclonal antibodies produced by HIC1 1-C10).Alternatively, a heterologous framework region, such as, but not limitedto a human framework region, can be included in the heavy and/or lightchain of the antibodies.

In one example the sequence of the specificity determining regions ofeach CDR (from any of the antibodies disclosed herein) is determined.Residues outside the SDR (non-ligand contacting sites) can besubstituted and the monoclonal antibody retains its ability to bindpancreatic endocrine cells. For example, in any of the CDR sequences asin the table above, at most one, two or three amino acids aresubstituted.

The production of chimeric antibodies, which include a framework regionfrom one antibody and the CDRs from a different antibody, is well knownin the art. For example, humanized antibodies can be routinely produced.The antibody or antibody fragment can be a humanized immunoglobulinhaving complementarity determining regions (CDRs) from a donormonoclonal antibody that binds a cell surface antigen of pancreaticcells (such as endocrine, exocrine or ductal cells) and immunoglobulinand heavy and light chain variable region frameworks from human acceptorimmunoglobulin heavy and light chain frameworks. Generally, thehumanized immunoglobulin specifically binds to the cell surface antigen(or cells expressing the antigen) with an affinity constant of at least10⁷ M⁻¹, such as at least 10⁸ M⁻¹ at least 5×10⁸ M⁻¹ or at least 10⁹M⁻¹.

Humanized monoclonal antibodies can be produced by transferring donorcomplementarity determining regions (CDRs) from heavy and light variablechains of the donor mouse immunoglobulin. In one example, the CDRs fromthe monoclonal antibodies produced by the hybridomas HIC0 4-F9, HIC12-B4, HIC1-4-G6, HIC1 7-H10, HIC0 3-05, HIC1 5-F10, DHIC2 2-B4, or DHIC22-C12 are transferred into a human variable domain, and thensubstituting human residues in the framework regions when required toretain affinity. In another example, the CDRs from the monoclonalantibodies produced by the hybridomas DHIC2 4-A10 or DHIC3 5-H10 aretransferred into a human variable domain, and then substituting humanresidues in the framework regions when required to retain affinity. In afurther example, the CDRs from the monoclonal antibody produced by thehybridoma HIC1 1-C10 are transferred into a human variable domain, andthen substituting human residues in the framework regions when requiredto retain affinity.

The use of antibody components derived from humanized monoclonalantibodies obviates potential problems associated with theimmunogenicity of the constant regions of the donor antibody. Techniquesfor producing humanized monoclonal antibodies are described, forexample, by Jones et al., Nature 321:522, 1986; Riechmann et al., Nature332:323, 1988; Verhoeyen et al., Science 239:1534, 1988; Carter et al.,Proc. Natl. Acad. Sci. U.S.A. 89:4285, 1992; Sandhu, Crit. Rev. Biotech.12:437, 1992; and Singer et al., J. Immunol. 150:2844, 1993. Theantibody may be of any isotype, but in several embodiments the antibodyis an IgM or an IgG, including but not limited to, IgG₁, IgG₂, IgG₃ andIgG₄.

In one embodiment, the sequence of the humanized immunoglobulin heavychain variable region framework can be at least about 65% identical tothe sequence of the donor immunoglobulin heavy chain variable regionframework. Thus, the sequence of the humanized immunoglobulin heavychain variable region framework can be at least about 75%, at leastabout 85%, at least about 95%, or at least about 99% identical to thesequence of the donor murine immunoglobulin heavy chain variable regionframework. Human framework regions, and mutations that can be made in ahumanized antibody framework regions, are known in the art (see, forexample, in U.S. Pat. No. 5,585,089, which is incorporated herein byreference). One of skill in the art can readily select a human frameworkregion of use.

Exemplary human antibodies are shown in FIG. 3; additional exemplaryhuman antibodies are LEN and 21/28 CL. The sequences of the heavy andlight chain frameworks are known in the art. Exemplary light chainframeworks of human MAb LEN have the following sequences:

FR1: DIVMTQS PDSLAVSLGERATINC (SEQ ID NO: 20) FR2: WYQQKPGQPPLLIY(SEQ ID NO: 21) FR3: GVPDRPFGSGSGTDFTLTISSLQAEDVAVYYC (SEQ ID NO: 22)FR4: FGQGQTKLEIK (SEQ ID NO: 23)

Exemplary heavy chain frameworks of human MAb 21/28′ CL have thefollowing sequences:

FR1: QVQLVQSGAEVKKPQASVKVSCKASQYTFT (SEQ ID NO: 24) FR2: WVRQAPGQRLEWMG(SEQ ID NO: 25) FR3: RVTITRDTSASTAYMELSSLRSEDTAVYYCAR (SEQ ID NO: 26)FR4: WGQGTLVTVSS. (SEQ ID NO: 27)These framework sequences are provided for example only; a humanizedantibody can include the human framework region from any humanmonoclonal antibody of interest. In one example, the human frameworkregion includes the amino acid sequence set forth as SEQ ID NO: 40 (seeFIG. 3C).

Antibodies, such as murine monoclonal antibodies, chimeric antibodies,and humanized antibodies, include full length molecules as well asfragments thereof, such as Fab, F(ab′)₂, and Fv which include a heavychain and light chain variable region and are capable of binding theepitopic determinant on pancreatic endocrine cells or a subset thereof,pancreatic exocrine cells, or pancreatic ductal cells. These antibodyfragments retain some ability to selectively bind with their antigen orreceptor. These fragments include:

(1) Fab, the fragment which contains a monovalent antigen-bindingfragment of an antibody molecule, can be produced by digestion of wholeantibody with the enzyme papain to yield an intact light chain and aportion of one heavy chain;

(2) Fab′, the fragment of an antibody molecule can be obtained bytreating whole antibody with pepsin, followed by reduction, to yield anintact light chain and a portion of the heavy chain; two Fab′ fragmentsare obtained per antibody molecule;

(3) (Fab′)₂, the fragment of the antibody that can be obtained bytreating whole antibody with the enzyme pepsin without subsequentreduction; F(ab′)₂ is a dimer of two Fab′ fragments held together by twodisulfide bonds;

(4) Fv, a genetically engineered fragment containing the variable regionof the light chain and the variable region of the heavy chain expressedas two chains; and

(5) Single chain antibody (such as scFv), defined as a geneticallyengineered molecule containing the variable region of the light chain,the variable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule.

(6) A dimer of a single chain antibody (scFV₂), defined as a dimer of anscFV. This has also been termed a “miniantibody.”

Methods of making these fragments are known in the art (see for example,Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, New York, 1988). In several examples, the variable regionincluded in the antibody is the variable region of the monoclonalantibody produced by the hybridoma HIC0 4-F9, HIC1 2-B4, HIC1 4-G6, HIC17-H10, HIC0 3-05, HIC1 5-F10, DHIC2 2-B4, or DHIC2 2-C12 (deposited inaccordance with the Budapest treaty on Apr. 27, 2006) or a progenythereof. In one group of embodiments, the antibodies have V_(H) CDRshaving the amino acid sequences shown above in Table 1, or a combinationof these CDRs, as discussed above. In another group of embodiments, theantibodies have VH and VL CDRs from one of the monoclonal antibodiesproduced by the hybridomas HIC0 4-F9, HIC1 2-B4, HIC1 4-G6, HIC1-7-H10,HIC0 3-05, HIC1 5-F10, DHIC2 2-B4, or DHIC2 2-C12 or a progeny thereof.Patent deposits of hybridomas HIC0 3-05, HIC0 4-F9, DHIC2 2-B4 and DHIC2-C12, HIC1 5-F10, HIC1 7-H10, HIC1 2-B4 and HIC1 4-G6 were made withthe ATCC on Apr. 27, 2006, in accordance with the Budapest Treaty(Accession Nos. PTA-7556, PTA-7557, PTA-7558, PTA-7559, PTA-7560,PTA-7561, PTA-7562 and PTA-7563, respectively. In a further group ofembodiments, the antibodies have VH and VL CDRs from one of themonoclonal antibodies produced by the hybridomas DHIC2 4-A10, DHIC35-H100R HIC1 1-C10 (deposited in accordance with the Budapest Treaty onApr. 26, 2007) or a progeny thereof.

In a further group of embodiments, the antibodies are Fv antibodies,which are typically about 25 kDa and contain a complete antigen-bindingsite with three CDRs per each heavy chain and each light chain. Toproduce these antibodies, the V_(H) and the V_(L) can be expressed fromtwo individual nucleic acid constructs in a host cell. If the V_(H) andthe V_(L) are expressed non-contiguously, the chains of the Fv antibodyare typically held together by noncovalent interactions. However, thesechains tend to dissociate upon dilution, so methods have been developedto crosslink the chains through glutaraldehyde, intermoleculardisulfides, or a peptide linker. Thus, in one example, the Fv can be adisulfide stabilized Fv (dsFv), wherein the heavy chain variable regionand the light chain variable region are chemically linked by disulfidebonds.

In an additional example, the Fv fragments comprise V_(H) and V_(L)chains connected by a peptide linker. These single-chain antigen bindingproteins (scFv) are prepared by constructing a structural genecomprising DNA sequences encoding the V_(H) and V_(L) domains connectedby an oligonucleotide. The structural gene is inserted into anexpression vector, which is subsequently introduced into a host cellsuch as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing scFvs are known in the art (see Whitlow et al.,Methods: a Companion to Methods in Enzymology, Vol. 2, page 97, 1991;Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack etal., Bio/Technology 11:1271, 1993; and Sandhu, supra). Dimers of asingle chain antibody (scFV₂), are also contemplated.

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli of DNA encoding the fragment.Antibody fragments can be obtained by pepsin or papain digestion ofwhole antibodies by conventional methods. For example, antibodyfragments can be produced by enzymatic cleavage of antibodies withpepsin to provide a 5S fragment denoted F(ab′)₂. This fragment can befurther cleaved using a thiol reducing agent, and optionally a blockinggroup for the sulfhydryl groups resulting from cleavage of disulfidelinkages, to produce 3.5S Fab′ monovalent fragments. Alternatively, anenzymatic cleavage using pepsin produces two monovalent Fab′ fragmentsand an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat.No. 4,331,647, and references contained therein; Nisonhoff et al., Arch.Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959;Edelman et al., Methods in Enzymology, Vol. 1, page 422, Academic Press,1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody.

One of skill will realize that conservative variants of the antibodiescan be produced. Such conservative variants employed in antibodyfragments, such as dsFv fragments or in scFv fragments, will retaincritical amino acid residues necessary for correct folding andstabilizing between the V_(H) and the V_(L) regions, and will retain thecharge characteristics of the residues in order to preserve the low pIand low toxicity of the molecules. Amino acid substitutions (such as atmost one, at most two, at most three, at most four, or at most fiveamino acid substitutions) can be made in the V_(H) and the V_(L) regionsto increase yield. Conservative amino acid substitution tables providingfunctionally similar amino acids are well known to one of ordinary skillin the art. The following six groups are examples of amino acids thatare considered to be conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

Thus, one of skill in the art can readily review the sequences shown inTable 1, identify a conservative substitution, and produce theconservative variant using well-known molecular techniques.

Effector molecules, such as therapeutic, diagnostic, or detectionmoieties can be linked to an antibody of interest, such as an antibodythat specifically binds a cell surface antigen on human pancreaticendocrine cells or a subset thereof, an antibody that specifically bindsa cell surface antigen on a pancreatic exocrine cell, or an antibodythat specifically binds a cell surface antigen on a pancreatic ductalcell, using any number of means known to those of skill in the art. Bothcovalent and noncovalent attachment means may be used. The procedure forattaching an effector molecule to an antibody varies according to thechemical structure of the effector. Polypeptides typically contain avariety of functional groups; such as carboxylic acid (COOH), free amine(—NH₂) or sulfhydryl (—SH) groups, which are available for reaction witha suitable functional group on an antibody to result in the binding ofthe effector molecule. Alternatively, the antibody is derivatized toexpose or attach additional reactive functional groups. Thederivatization may involve attachment of any of a number of linkermolecules such as those available from Pierce Chemical Company,Rockford, Ill. The linker can be any molecule used to join the antibodyto the effector molecule. The linker is capable of forming covalentbonds to both the antibody and to the effector molecule. Suitablelinkers are well known to those of skill in the art and include, but arenot limited to, straight or branched-chain carbon linkers, heterocycliccarbon linkers, or peptide linkers. Where the antibody and the effectormolecule are polypeptides, the linkers may be joined to the constituentamino acids through their side groups (such as through a disulfidelinkage to cysteine) or to the alpha carbon amino and carboxyl groups ofthe terminal amino acids.

In some circumstances, it is desirable to free the effector moleculefrom the antibody when the immunoconjugate has reached its target site.Therefore, in these circumstances, immunoconjugates will compriselinkages that are cleavable in the vicinity of the target site. Cleavageof the linker to release the effector molecule from the antibody may beprompted by enzymatic activity or conditions to which theimmunoconjugate is subjected either inside the target cell or in thevicinity of the target site. When the target site is a tumor, a linkerwhich is cleavable under conditions present at the tumor site (forexample, when exposed to tumor-associated enzymes or acidic pH) may beused.

In view of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, label (such as enzymes or fluorescent molecules) drugs,toxins, and other agents to antibodies one skilled in the art will beable to determine a suitable method for attaching a given agent to anantibody or other polypeptide.

Therapeutic agents include various drugs such as vinblastine, daunomycinand the like, and effector molecules such as cytotoxins including butnot limited to native or modified Pseudomonas exotoxin or Diphtheriatoxin, encapsulating agents, (such as liposomes) which themselvescontain pharmacological compositions, target moieties and ligands. Thechoice of a particular therapeutic agent depends on the particulartarget molecule or cell and the biological effect desired to be evoked.Thus, for example, the therapeutic agent may be an effector moleculethat is a cytotoxin which is used to bring about the death of aparticular target cell, such as a pancreatic endocrine cells tumor (suchas an insulinoma or glucagonoma). Conversely, where a non-lethalbiological response is desired, a therapeutic agent can be conjugated toa non-lethal pharmacological agent or a liposome containing a non-lethalpharmacological agent.

Toxins can be employed with antibodies that specifically bind a cellsurface antigen of a human pancreatic endocrine cells or a subsetthereof, and fragments of these antibodies, for example, a svFv or adsFv, to yield chimeric molecules, which are of use as immunotoxins.Exemplary toxins include Pseudomonas exotoxin (PE), ricin, abrin,diphtheria toxin and subunits thereof, ribotoxin, ribonuclease, saporin,gelonin and calicheamicin, as well as botulinum toxins A through F.These toxins are well known in the art and many are readily availablefrom commercial sources (for example, Sigma Chemical Company, St. Louis,Mo.). These antibodies are of use, for example, for the treatment oftumors of pancreatic endocrine cells, such as insulinomas.

Diphtheria toxin is isolated from Corynebacterium diphtheriae.Typically, diphtheria toxin for use in immunotoxins is mutated to reduceor to eliminate non-specific toxicity. A mutant known as CRM107, whichhas full enzymatic activity but markedly reduced non-specific toxicity,has been known since the 1970's (Laird and Groman, J. Virol. 19:220,1976), and has been used in human clinical trials. See, U.S. Pat. No.5,792,458 and U.S. Pat. No. 5,208,021.

Ricin is the lectin RCA60 from Ricinus communis (Castor bean). Forexamples of ricin, see, U.S. Pat. No. 5,079,163 and U.S. Pat. No.4,689,401. Ricinus communis agglutinin (RCA) occurs in two formsdesignated RCA₆₀ and RCA₁₂₀ according to their molecular weights ofapproximately 65 and 120 kD, respectively (Nicholson & Blaustein, J.Biochim. Biophys. Acta 266:543, 1972). The A chain is responsible forinactivating protein synthesis and killing cells. The B chain bindsricin to cell-surface galactose residues and facilitates transport ofthe A chain into the cytosol (Olsnes et al., Nature 249:627-631, 1974and U.S. Pat. No. 3,060,165).

Ribonucleases have also been conjugated to targeting molecules for useas immunotoxins (see Suzuki et al., Nat. Biotech. 17:265-70, 1999).Exemplary ribotoxins such as α-sarcin and restrictocin are discussed in,for example Rathore et al., Gene 190:31-5, 1997; and Goyal and Batra,Biochem. 345 Pt 2:247-54, 2000. Calicheamicins were first isolated fromMicromonospora echinospora and are members of the enediyne antitumorantibiotic family that cause double strand breaks in DNA that lead toapoptosis (see, for example Lee et al., J. Antibiot. 42:1070-87, 1989).The drug is the toxic moiety of an immunotoxin in clinical trials (see,for example, Gillespie et al., Ann. Oncol. 11:735-41, 2000).

Abrin includes toxic lectins from Abrus precatorius. The toxicprinciples, abrin a, b, c, and d, have a molecular weight of from about63 and 67 kD and are composed of two disulfide-linked polypeptide chainsA and B. The A chain inhibits protein synthesis; the B chain (abrin-b)binds to D-galactose residues (see, Funatsu et al., Agr. Biol. Chem.52:1095, 1988; and Olsnes, Methods Enzymol. 50:330-335, 1978).

In one embodiment, the toxin is Pseudomonas exotoxin (PE). NativePseudomonas exotoxin A (“PE”) is an extremely active monomeric protein(molecular weight 66 kD), secreted by Pseudomonas aeruginosa, whichinhibits protein synthesis in eukaryotic cells. The native PE sequenceand the sequence of modified PE are provided in U.S. Pat. No. 5,602,095,incorporated herein by reference. In one embodiment, native PE has asequence set forth as:

(SEQ ID NO: 28) AEEAFDLWNE CAKACVLDLK DGVRSSRMSV DPAIADTNGQGVLHYSMVLE GGNDALKLAI DNALSITSDG LTIRLEGGVEPNKPVRYSYT RQARGSWSLN WLVPIGHEKP SNIKVFIHELNAGNQLSHMS PIYTIEMGDE LLAKLARDAT FFVRAHESNEMQPTLAISHA GVSVVMAQTQ PRREKRWSEW ASGKVLCLLDPLDGVYNYLA QQRCNLDDTW EGKIYRVLAG NPAKHDLDIKPTVISHRLHF PEGGSLAALT AHQACHLPLE TFTRHRQPRGWEQLEQCGYP VQRLVALYLA ARLSWNQVDQ VIRNALASPGSGGDLGEAIR EQPEQARLAL TLAAAESERF VRQGTGNDEAGAANADVVSL TCPVAAGECA GPADSGDALL ERNYPTGAEFLGDGGDVSFS TRGTQNWTVE RLLQAHRQLE ERGYVFVGYHGTFLEAAQSI VFGGVRARSQ DLDAIWRGFY IAGDPALAYGYAQDQEPDAR GRIRNGALLR VYVPRSSLPG FYRTSLTLAAPEAAGEVERL IGHPLPLRLD AITGPEEEGG RLETILGWPLAERTVVIPSA IPTDPRNVGG DLDPSSIPDK EQAISALPDY ASQPGKPPRE DLK

Thus, the PE used in the immunotoxins disclosed herein includes thenative sequence, cytotoxic fragments of the native sequence, andconservatively modified variants of native PE and its cytotoxicfragments. Cytotoxic fragments of PE include those which are cytotoxicwith or without subsequent proteolytic or other processing in the targetcell (such as a protein or pre-protein). Cytotoxic fragments of PE knownin the art include PE40, PE38, and PE35. In several embodiments, the PEhas been modified to reduce or eliminate non-specific cell binding,typically by deleting domain Ia, as taught in U.S. Pat. No. 4,892,827.U.S. Pat. No. 5,512,658, for instance, discloses that a mutated PE inwhich Domain Ia is present but in which the basic residues of domain Iaat positions 57, 246, 247, and 249 are replaced with acidic residues(glutamic acid, or “E”) exhibits greatly diminished non-specificcytotoxicity. PE40 is a truncated derivative of PE (see, Pai et al.,Proc. Natl. Acad. Sci. USA 88:3358-62, 1991; and Kondo et al., J. Biol.Chem. 263:9470-9475, 1988).

The antibodies or antibody fragments disclosed herein can be derivatizedor linked to another molecule (such as another peptide or protein). Ingeneral, the antibodies or portion thereof is derivatized such that thebinding to pancreatic endocrine cells is not affected adversely by thederivatization or labeling. For example, the antibody can befunctionally linked (by chemical coupling, genetic fusion, noncovalentassociation or otherwise) to one or more other molecular entities, suchas another antibody (for example, a bispecific antibody or a diabody), adetection agent, a pharmaceutical agent, and/or a protein or peptidethat can mediate association of the antibody or antibody portion withanother molecule (such as a streptavidin core region or a polyhistidinetag).

One type of derivatized antibody is produced by crosslinking two or moreantibodies (of the same type or of different types, such as to createbispecific antibodies). Suitable crosslinkers include those that areheterobifunctional, having two distinctly reactive groups separated byan appropriate spacer (such as m-maleimidobenzoyl-N-hydroxysuccinimideester) or homobifunctional (such as disuccinimidyl suberate). Suchlinkers are available from Pierce Chemical Company, Rockford, Ill.

An antibody that binds pancreatic endocrine cells or a subset thereof,pancreatic exocrine cells or a subset thereof, or pancreatic ductalcells or a subset thereof, can also be labeled. Useful detection agentsinclude fluorescent compounds, including fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin, lanthanide phosphors and the like.Bioluminescent markers are also of use, such as Green fluorescentprotein (GFP), Yellow fluorescent protein (YFP) and enhanced variants ofthese proteins. An antibody can also be labeled with enzymes that areuseful for detection, such as horseradish peroxidase, β-galactosidase,luciferase, alkaline phosphatase, glucose oxidase and the like. When anantibody is labeled with a detectable enzyme, it can be detected byadding additional reagents that the enzyme uses to produce a reactionproduct that can be discerned. For example, when the agent horseradishperoxidase is present, the addition of hydrogen peroxide anddiaminobenzidine leads to a colored reaction product, which is visuallydetectable. An antibody may also be labeled with biotin, and detectedthrough indirect measurement of avidin or streptavidin binding. Itshould be noted that the avidin itself can be labeled with an enzyme ora fluorescent label.

An antibody may be labeled with a paramagnetic agent, such asgadolinium. Antibodies can also be labeled with lanthanides (such aseuropium and dysprosium), and manganese. Paramagnetic particles such assuperparamagnetic iron oxide are also of use as labels. An antibody mayalso be labeled with a predetermined polypeptide epitopes recognized bya secondary reporter (such as leucine zipper pair sequences, bindingsites for secondary antibodies, metal binding domains, epitope tags). Insome embodiments, labels are attached by spacer arms of various lengthsto reduce steric hindrance.

An antibody can also be labeled with a radiolabeled amino acid. Theradiolabel may be used for both diagnostic and therapeutic purposes. Forinstance, the radiolabel may be used to detect pancreatic endocrinecells or a subset thereof, pancreatic exocrine cells or a subsetthereof, pancreatic ductal cells or a subset thereof, or a pancreatictumor, by x-ray or other diagnostic techniques. Further, the radiolabelmay be used therapeutically as a toxin for islet cell tumors or apancreatic adenocarcinoma. Examples of labels for polypeptides include,but are not limited to, the following radioisotopes or radionuclides:³H, ¹⁴C, C and ¹²⁵I.

An antibody can also be derivatized with a chemical group such aspolyethylene glycol (PEG), a methyl or ethyl group, or a carbohydrategroup. These groups may be useful to improve the biologicalcharacteristics of the antibody, such as to increase serum half-life orto increase tissue binding.

Nucleic acids encoding the amino acid sequences of the antibodies thatbind pancreatic endocrine cells or a subset thereof are also providedherein. Nucleic acids encoding antibodies including one or more CDRsshown in Table 1, nucleic acids encoding antibodies that have thepolypeptide sequence of the antibodies produced by a HIC0 4-F9, HIC12-B4, HIC1 4-G6, HIC1 7-H10, HIC0 3-05, HIC1-5-F10, DHIC2 2-B4, or DHIC22-C12 hybridoma; DHIC2 4-A10 or DHIC3 5-H10 hybridoma; or a HIC1 1-C10hybridoma (or a humanized form of any of these antibodies) can readilybe produced by one of skill in the art.

Exemplary nucleic acid sequences are as follows:

TABLE 3 Hybridoma ID Nucleotide sequence for heavy chain V-regionHIC0-4F9 CAGAAGTTCAGCTGCAGCAGTCTGGGGCTGAGCTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAATATTAAAGACTACTATATACACTGGGTGAAGCAGAAGCCTGAACAGGGCCTGGAGTGGATTGGATGGATTGATCCTGAGAATGGTAATACTATATATGACCCGAAGTTCCAGGACAAGGCCAGTATAACTTCAGACACATCCTCCAACACAGCCTACCTCCAGCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTACTAGTTACTACGGTAGTACCTACTACTTTGACTACTGGGGCCAAGGCACCA (SEQ ID NO: 29) DHIC2 2-ATGGCGGCGGCGCAGAGCATTCAGGTGCAGATTCAGCTGGTGCA C12GAGCGGCCCGGAACTGAAAAAACCGGGCGAAACCGTGAAAATTAGCTGCAAAGCGAGCGGCTATACCTTTACCGATTATAGCATGCATTGGGTGAAACAGGCGCCGGGCAAAGGCCTGAAATGGATGGGCTGGATTAACACCGAAACCGGCGAACCGACCTATGCGGATGATTTTAAAGGCCGCTTTGCGTTTAGCCTGGAAACCAGCGCGAGCACCGCGTATCTGCAGATTAACAACCTGAAAAACGAAGATACCGCGACCTATTTTTGCAGCCGCGGCTATGGCAGCAGCAGCTGGTTTGCGTATTGGGGCCAGGGCACCCTGGTGACCGTGAGCGCGGAAAGCCAGA GCTTTCCGAAC (SEQ ID NO: 30)HIC0 3-05 TGAAGTGATGCTGGTGGAGTCTGGGGGAGGCTTAGTGAAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACTATGCCATGTCTTGGGTTCGCCAGACTCCGGAGAAGAGGCTGGAGTGGGTCGCAACCATTAGTAGTGGTGGTAGTTACACCTACTATCCAGACAGTGTGAAGGGGCGATTCACCATCTCCAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCAGTCTGAGGTCTGAGGACACGGCCATGTATTACTGTGCAAGACAGGGGGATAACTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCAGAGAGTCAGTC (SEQ ID NO: 31) HIC1 5-GAGTGGTGCCTTGGCCCCAATAATCAAAATACTTCTCGTGTCTTG F10CACAGAAATAGACCGCAGAGTCTTCAGATGTCAATCTACTAAGTTCCATATAGACTGTGCTGGAGGATTTGTCCGCAGTCAATGTGGCCTTGTCCTTGAATTTCTCACTGTACTTTAGACCACCACTTCCAGGGTAAAACCACCCAATCCACTCAAGACCCTGTCCAGACCTCTGCTTTACCCAGTGTATAATATACTCAGTGAAGGTGTAGCCAGAAGCCTTGCAGGACAGCTTCACTGATGCCCCGGGTTTCACCAGCTCAGCTCCAGACTGCTGCAGCTGGAC (SEQ ID NO: 32) HIC1 4-G6TGAGGTGAAGCTGGTGGAGTCTGGAGGAGGCTTGGTACAGCCTGGGGGTTCTCTGAGACTCTCCTGTGCAACTTCTGGATTCACCTTCACTGATTACTACATGCACTGGGTCCGCCAGCCTCCAGGAAAGGCACTTGAGTGGTTGGGTTTTATTAGAAACAAAGCTAATGGTTACACAACAGAGTACAGTGCATCTGTGAAGGGTCGGTTCACCATCTCCAGAGATAATTCCCAAAGCATCCTCTATCTTCAAATGAACACCCTGAGAGCTGAGGACAGTGCCACTTATTACTGTACAAGAGATATAAAGGGGGACTACTGGGGTCAAGGAACCTCAGT (SEQ ID NO: 33) HIC1 7-GAGTCCCTTGGCCCCAATAAGCAAACCAGGGTTGGTTACCCCCA H10TGTCTTGTACAGTAATACATGGCTGTGTCTTCAGACCTCAGACTGCTCATCTGCAGGGACAGGGTGTTCTTGGCATTGTCTCTGGAAATGGTGAATCGGCCCTTCACAGTGTTTGGAAAGTAGGTGCTACCACCACCACTACTAATGTATGCGACCCACTCCAGCCTCTTGTCCGGAGCCTGGCGAACCCAAGACATGTCATAGCTACTGAAAGCGAATCCAGAGGCTGCACAGGAGAGTTTCAGGGACCCTCCAGGCTTCACTAAGCCTCCCCCAGACTCCACCAGTTGCACTTCA (SEQ ID NO: 34)

Nucleotides molecules encoding the antibodies can readily be produced byone of skill in the art, using the amino acid sequences provided herein,and the genetic code. In addition, one of skill can readily construct avariety of clones containing functionally equivalent nucleic acids, suchas nucleic acids which differ in sequence but which encode the sameeffector molecule (“EM”) or antibody sequence. Thus, nucleic acidsencoding antibodies, conjugates and fusion proteins are provided herein.

Nucleic acid sequences encoding the antibodies that specifically bindpancreatic endocrine cells or a subset thereof, nucleic acid sequencesencoding the antibodies that specifically bind pancreatic exocrinecells, and nucleic acid sequences encoding the antibodies thatspecifically bind pancreatic ductal cells can be prepared by anysuitable method including, for example, cloning of appropriate sequencesor by direct chemical synthesis by methods such as the phosphotriestermethod of Narang et al., Meth. Enzymol. 68:90-99, 1979; thephosphodiester method of Brown et al., Meth. Enzymol. 68:109-151, 1979;the diethylphosphoramidite method of Beaucage et al., Tetra. Lett.22:1859-1862, 1981; the solid phase phosphoramidite triester methoddescribed by Beaucage & Caruthers, Tetra. Letts. 22(20):1859-1862, 1981,for example, using an automated synthesizer as described in, forexample, Needham-VanDevanter et al., Nucl. Acids Res. 12:6159-6168,1984; and, the solid support method of U.S. Pat. No. 4,458,066. Chemicalsynthesis produces a single stranded oligonucleotide. This can beconverted into double stranded DNA by hybridization with a complementarysequence, or by polymerization with a DNA polymerase using the singlestrand as a template. One of skill would recognize that while chemicalsynthesis of DNA is generally limited to sequences of about 100 bases,longer sequences may be obtained by the ligation of shorter sequences.

Exemplary nucleic acids encoding sequences encoding an antibody thatspecifically binds pancreatic endocrine cells or a subset thereof, anantibody that specifically binds pancreatic exocrine cells or a subsetthereof, or an antibody that specifically binds pancreatic ductal cellsor a subset thereof can be prepared by cloning techniques. Examples ofappropriate cloning and sequencing techniques, and instructionssufficient to direct persons of skill through cloning are found inSambrook et al., supra, Berger and Kimmel (eds.), supra, and Ausubel,supra. Product information from manufacturers of biological reagents andexperimental equipment also provide useful information. Suchmanufacturers include the SIGMA Chemical Company (Saint Louis, Mo.), R&DSystems (Minneapolis, Minn.), Pharmacia Amersham (Piscataway, N.J.),CLONTECH Laboratories, Inc. (Palo Alto, Calif.), Chem Genes Corp.,Aldrich Chemical Company (Milwaukee, Wis.), Glen Research, Inc., GIBCOBRL Life Technologies, Inc. (Gaithersburg, Md.), FlukaChemica-Biochemika Analytika (Fluka Chemie AG, Buchs, Switzerland),Invitrogen (San Diego, Calif.), and Applied Biosystems (Foster City,Calif.), as well as many other commercial sources known to one of skill

Nucleic acids can also be prepared by amplification methods.Amplification methods include polymerase chain reaction (PCR), theligase chain reaction (LCR), the transcription-based amplificationsystem (TAS), the self-sustained sequence replication system (3SR). Awide variety of cloning methods, host cells, and in vitro amplificationmethodologies are well known to persons of skill

In one example, an antibody of use is prepared by inserting the cDNAwhich encodes a variable region from an antibody into a vector whichcomprises the cDNA encoding an effector molecule (EM), such as an enzymeor label. The insertion is made so that the variable region and the EMare read in frame so that one continuous polypeptide is produced. Thus,the encoded polypeptide contains a functional Fv region and a functionalEM region. In one embodiment, cDNA encoding an enzyme is ligated to ascFv so that the enzyme is located at the carboxyl terminus of the scFv.In several examples, cDNA encoding a horseradish peroxidase or alkalinephosphatase, or a polypeptide marker of interest is ligated to a scFv sothat the enzyme (or polypeptide marker) is located at the amino terminusof the scFv. In another example, the label is located at the aminoterminus of the scFv. In a further example, cDNA encoding the protein orpolypeptide marker is ligated to a heavy chain variable region of anantibody, so that the enzyme or polypeptide marker is located at thecarboxyl terminus of the heavy chain variable region. The heavychain-variable region can subsequently be ligated to a light chainvariable region of the antibody using disulfide bonds. In a yet anotherexample, cDNA encoding an enzyme or a polypeptide marker is ligated to alight chain variable region of an antibody, so that the enzyme orpolypeptide marker is located at the carboxyl terminus of the lightchain variable region. The light chain-variable region can subsequentlybe ligated to a heavy chain variable region of the antibody usingdisulfide bonds.

Once the nucleic acids encoding the immunotoxin, antibody, labeledantibody, or fragment thereof are isolated and cloned, the protein canbe expressed in a recombinantly engineered cell such as bacteria, plant,yeast, insect and mammalian cells using a suitable expression vector.One or more DNA sequences encoding the antibody or fragment thereof canbe expressed in vitro by DNA transfer into a suitable host cell. Thecell may be prokaryotic or eukaryotic. The term also includes anyprogeny of the subject host cell. It is understood that all progeny maynot be identical to the parental cell since there may be mutations thatoccur during replication. Methods of stable transfer, meaning that theforeign DNA is continuously maintained in the host, are known in theart.

Polynucleotide sequences encoding the antibody, labeled antibody, orfunctional fragment thereof, can be operatively linked to expressioncontrol sequences. An expression control sequence operatively linked toa coding sequence is ligated such that expression of the coding sequenceis achieved under conditions compatible with the expression controlsequences. The expression control sequences include, but are not limitedto appropriate promoters, enhancers, transcription terminators, a startcodon (i.e., ATG) in front of a protein-encoding gene, splicing signalfor introns, maintenance of the correct reading frame of that gene topermit proper translation of mRNA, and stop codons.

The polynucleotide sequences encoding the antibody, labeled antibody, orfunctional fragment thereof can be inserted into an expression vectorincluding, but not limited to a plasmid, virus or other vehicle that canbe manipulated to allow insertion or incorporation of sequences and canbe expressed in either prokaryotes or eukaryotes. Hosts can includemicrobial, yeast, insect and mammalian organisms. Methods of expressingDNA sequences having eukaryotic or viral sequences in prokaryotes arewell known in the art. Biologically functional viral and plasmid DNAvectors capable of expression and replication in a host are known in theart.

Transformation of a host cell with recombinant DNA may be carried out byconventional techniques as are well known to those skilled in the art.Where the host is prokaryotic, such as E. coli, competent cells whichare capable of DNA uptake can be prepared from cells harvested afterexponential growth phase and subsequently treated by the CaCl₂ methodusing procedures well known in the art. Alternatively, MgCl₂ or RbC1 canbe used. Transformation can also be performed after forming a protoplastof the host cell if desired, or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate coprecipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin liposomes, or virus vectors may be used. Eukaryotic cells can also becotransformed with polynucleotide sequences encoding the antibody,labeled antibody, or functional fragment thereof, and a second foreignDNA molecule encoding a selectable phenotype, such as the herpes simplexthymidine kinase gene. Another method is to use a eukaryotic viralvector, such as simian virus 40 (SV40) or bovine papilloma virus, totransiently infect or transform eukaryotic cells and express the protein(see for example, Eukaryotic Viral Vectors, Cold Spring HarborLaboratory, Gluzman ed., 1982). One of skill in the art can readily usean expression systems such as plasmids and vectors of use in producingproteins in cells including higher eukaryotic cells such as the COS,CHO, HeLa and myeloma cell lines.

Isolation and purification of recombinantly expressed polypeptide can becarried out by conventional means including preparative chromatographyand immunological separations. Once expressed, the antibody, labeledantibody or functional fragment thereof can be purified according tostandard procedures of the art, including ammonium sulfateprecipitation, affinity columns, column chromatography, and the like(see, generally, R. Scopes, Protein Purification, Springer-Verlag, N.Y.,1982). Substantially pure compositions of at least about 90 to 95%homogeneity are disclosed herein, and 98 to 99% or more homogeneity canbe used for pharmaceutical purposes. Once purified, partially or tohomogeneity as desired, if to be used therapeutically, the polypeptidesshould be substantially free of endotoxin.

Methods for expression of single chain antibodies and/or refolding to anappropriate active form, including single chain antibodies, frombacteria such as E. coli have been described and are well-known and areapplicable to the antibodies disclosed herein. See, Buchner et al.,Anal. Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991;Huse et al., Science 246:1275, 1989 and Ward et al., Nature 341:544,1989, all incorporated by reference herein.

Often, functional heterologous proteins from E. coli or other bacteriaare isolated from inclusion bodies and require solubilization usingstrong denaturants, and subsequent refolding. During the solubilizationstep, as is well known in the art, a reducing agent must be present toseparate disulfide bonds. An exemplary buffer with a reducing agent is:0.1 M Tris pH 8, 6 M guanidine, 2 mM EDTA, 0.3 M DTE (dithioerythritol).Reoxidation of the disulfide bonds can occur in the presence of lowmolecular weight thiol reagents in reduced and oxidized form, asdescribed in Saxena et al., Biochemistry 9: 5015-5021, 1970,incorporated by reference herein, and especially as described by Buchneret al., supra.

Renaturation is typically accomplished by dilution (for example,100-fold) of the denatured and reduced protein into refolding buffer. Anexemplary buffer is 0.1 M Tris, pH 8.0, 0.5 M L-arginine, 8 mM oxidizedglutathione (GSSG), and 2 mM EDTA.

As a modification to the two chain antibody purification protocol, theheavy and light chain regions are separately solubilized and reduced andthen combined in the refolding solution. An exemplary yield is obtainedwhen these two proteins are mixed in a molar ratio such that a 5 foldmolar excess of one protein over the other is not exceeded. Excessoxidized glutathione or other oxidizing low molecular weight compoundscan be added to the refolding solution after the redox-shuffling iscompleted.

In addition to recombinant methods, the antibodies, labeled antibodiesand functional fragments thereof that are disclosed herein can also beconstructed in whole or in part using standard peptide synthesis. Solidphase synthesis of the polypeptides of less than about 50 amino acids inlength can be accomplished by attaching the C-terminal amino acid of thesequence to an insoluble support followed by sequential addition of theremaining amino acids in the sequence. Techniques for solid phasesynthesis are described by Barany & Merrifield, The Peptides: Analysis,Synthesis, Biology. Vol. 2: Special Methods in Peptide Synthesis, PartA. pp. 3-284; Merrifield et al., J. Am. Chem. Soc. 85:2149-2156, 1963,and Stewart et al., Solid Phase Peptide Synthesis, 2nd ed., Pierce Chem.Co., Rockford, Ill., 1984. Proteins of greater length may be synthesizedby condensation of the amino and carboxyl termini of shorter fragments.Methods of forming peptide bonds by activation of a carboxyl terminalend (such as by the use of the coupling reagentN,N′-dicylohexylcarbodimide) are well known in the art.

Methods of Detection and Isolation

A method is provided herein for the detection and/or isolation ofpancreatic endocrine cells or a subset thereof from a biological sampleor cells in vitro. These methods include contacting cells with one ormore of the antibodies disclosed herein that specifically bind a cellsurface antigen on pancreatic endocrine cells to form an immune complex.The presence (or absence) of the immune complex is then detected and/orused to isolate cells of interest. Pancreatic endocrine cells can bedetected either in vivo or in vitro. Thus, in one example, the methodsdisclosed herein can be used to detect the number or mass of pancreaticendocrine cells (or a subset thereof) in a subject, such as a humansubject.

In one embodiment a reduction in the number or mass of pancreaticendocrine cells, as compared to a control, indicates that the subjecthas or is at risk of having diabetes. The number and or mass of alltypes of pancreatic endocrine cells, or a specific subset, such as theinsulin producing cells, can be determined using the methods disclosedherein. A control can be a standard value, or the number or mass ofpancreatic endocrine cells in a sample from a subject not afflicted withdiabetes, or the number or mass of pancreatic endocrine cells in asubject not afflicted with diabetes. In another embodiment, an increasein the number or mass of pancreatic endocrine cells, as compared to acontrol, indicates that the subject has a pancreatic endocrine celltumor. Thus, an increase in the number of pancreatic endocrine cells, orthe pancreatic endocrine cell mass, as compared to a subject without apancreatic endocrine cell tumor, indicates that the subject has, or isat risk of having, a pancreatic endocrine cell tumor. In a furtherembodiment, the method is used to indicate if a therapeutic treatment iseffective in a subject. Thus, in one example, the therapy is designed toincrease or maintain the number (or mass) of pancreatic endocrine cells,as compared to a subject without treatment. Maintenance of pancreaticendocrine cell tissue, or an increase in the number of pancreaticendocrine cells, as compared to a control, indicates that the treatmentis effective. In another example, the therapy is designed to decreasethe number (or mass) of pancreatic endocrine cells, such as for tumortreatment. A decrease in the number of pancreatic endocrine cells in thetumor, or a decrease in tumor mass, indicates that the treatment iseffective.

Methods are also provided herein for the detection of pancreaticexocrine cells. A method is provided herein for the detection and/orisolation of pancreatic exocrine cells or a subset thereof from abiological sample or cells in vitro. These methods include contactingcells with one or more of the antibodies disclosed herein to form animmune complex. The presence (or absence) of the immune complex is thendetected and/or used to isolate cells of interest. Pancreatic exocrinecells can be detected either in vivo or in vitro. Thus, in one example,the methods disclosed herein can be used to detect the number or mass ofpancreatic exocrine cells (or a subset thereof) in a subject, such as ahuman subject.

In one example, the methods are used to detect a pancreatic exocrinecell tumor. The number and or mass of pancreatic exocrine cells can bedetermined using the methods disclosed herein. In one embodiment, anincrease in the number or mass of pancreatic exocrine cells, as comparedto a control, indicates that the subject has a pancreatic exocrine celltumor. A control can be a standard value, or the number or mass ofpancreatic exocrine cells in a sample from a subject not afflicted witha tumor. Thus, an increase in the number of pancreatic exocrine cells,or the pancreatic exocrine cell mass, as compared to a subject without apancreatic exocrine cell tumor, indicates that the subject has, or is atrisk of having, a pancreatic exoncrine cell tumor.

A method is also provided herein for detecting pancreatic ductal cells.In one example, methods are provided for detecting a pancreaticadenocarcinoma. A method is provided herein for the detection and/orisolation of pancreatic ductal cells or a subset thereof from abiological sample or cells in vitro. These methods include contactingcells with the antibodies disclosed herein that specifically bindpancreatic ductal cells to form an immune complex. The presence (orabsence) of the immune complex is then detected and/or used to isolatecells of interest. Pancreatic ductal cells can be detected either invivo or in vitro. Thus, in one example, the methods disclosed herein canbe used to detect the number or mass of pancreatic ductal cells (or asubset thereof) in a subject, such as a human subject.

In one example, the methods are used to detect a pancreaticadenocarcinoma. The number and or mass of pancreatic ductal cells can bedetermined using the methods disclosed herein. In one embodiment, anincrease in the number or mass of pancreatic ductal cells, as comparedto a control, indicates that the subject has a pancreaticadenocarcinoma. A control can be a standard value, or the number or massof pancreatic ductal cells in a sample from a subject not afflicted withan adenocarcinoma. Thus, an increase in the number of pancreatic ductalcells, or the pancreatic ductal cell mass, as compared to a subjectwithout a pancreatic adenocarinoma, indicates that the subject has, oris at risk of having, a pancreatic adenocarcinoma.

In one embodiment, a sample is obtained from a subject, and the presenceof pancreatic endocrine cells, or pancreatic endocrine cell subset, isassessed in vitro. In another embodiment, a sample is obtained from asubject, and the presence of pancreatic exocrine cells, or pancreaticexocrine cell subset, is assessed in vitro. In a further embodiment, asample is obtained from a subject, and the presence of pancreatic ductalcells is assessed in vitro. A biological sample for in vitro testing istypically obtained from a mammalian subject of interest, such as a rat,mouse, cow, dog, guinea pig, rabbit, or primate, such as a non-human ora human primate. In one embodiment, the primate is macaque, chimpanzee,or a human.

In a further embodiment, when assessing pancreatic endocrine cells, thesubject has, is suspected of having, or is at risk of developingdiabetes. In another embodiment, when assessing pancreatic endocrinecells, the subject has, is suspected of having, or is at risk of havinga tumor of the pancreatic endocrine cells, such as an insulinoma. In yetanother embodiment, when assessing pancreatic exocrine cells, thesubject has, is suspected of having, or is at risk of having, apancreatic exocrine cell tumor. In an additional embodiment, whenassessing pancreatic ductal cells, the subject has, is suspected ofhaving, or is at risk of having a pancreatic adenocarcinoma.

The sample can be any sample, including, but not limited to, tissue frombiopsies, autopsies and pathology specimens. Biological samples alsoinclude sections of tissues, for example, frozen sections taken forhistological purposes. If desired, the antigen of interest can also bedetected in additional biological samples. Biological samples furtherinclude body fluids, such as blood, serum, spinal fluid, pancreaticductal fluid or urine.

The antibodies described herein can be used in immunohistochemicalassays. These assays are well known to one of skill in the art (seeHarlow & Lane, Antibodies, A Laboratory Manual, Cold Spring HarborPublications, New York (1988), for a description of immunoassayformats). The antibodies can also be used for fluorescence activatedcell sorting (FACS). A FACS employs a plurality of color channels, lowangle and obtuse light-scattering detection channels, and impedancechannels, among other more sophisticated levels of detection, toseparate or sort cells (see U.S. Pat. No. 5,061,620). The antibodies canalso be used for magnetic separation of endocrine cells, exocrine cellsand/or ductal cells. Magnetic separation involves the use ofparamagnetic particles which are: 1) conjugated to the pancreaticspecific antibodies, such as endocrine cell specific antibodies,exocrine cell antibodies and/or ductal cell antibodies; 2) conjugated todetection antibodies which are able to bind to the pancreatic specificantibodies; or 3) conjugated to a detection reagent (such as avidin)which can bind to detection antibodies (such as biotinylatedantibodies). Any of the antibodies disclosed herein can be used in theseassays. The antibodies can be used in methods that utilize positiveselectin (expressing the antigen of interest), negative selection (notexpressing the antigen of interest), or both (expressing one antigen ofinterest and not expressing a second antigen of interest).

An application of the antibodies described herein is the assessment ofpurity and/or functionality of a clinically-relevant pancreaticpopulation, such as an islet cell preparation, obtained from a cadavericdonor. Prior to transplantation to a human recipient, the extent oflabeling of intact islets or dissociated islet cells by these antibodiescan provide a useful measure of their quality and suitability fortransplantation. This extent of labeling could be determined by flowcytometry, immunocytochemistry, or by protein recognition in an ELISAassay.

In some embodiments, methods are provided for isolating pancreaticendocrine cells, a specific type of pancreatic endocrine cells, cellsexhibiting glucose-dependent insulin secretion, or islets of Langerhans,using the antibodies disclosed herein. Protocols for the isolation ofislets are well known in the art, see for example, Kinasiewicz et al.,Physiol. Res. 53: 327-333, 2004. These protocols can include bothpositive selection (for pancreatic endocrine cells) and negativeselection (so that exocrine cells and/or ductal cells are excluded).Thus, in several examples, the antibodies disclosed herein can be usedin the isolation of pancreatic endocrine cells, or a subset thereof,from the pancreas. However, pancreatic endocrine cells also can beisolated from culture systems designed to produce pancreatic endocrinecells. For example, the production of endocrine cells from ES cells isdescribed in PCT Application No. PCT/US02/02361. Pancreatic endocrinecells can also be isolated from proliferating cells of human cadavericislets or other pancreatic tissue in vitro. In one embodiment, theepithelial cells migrate out from the islets of Langerhans when culturedex vivo and undergo an epithelial to mesenchymal transition to form apopulation of mesenchymal cells (see Gershengorn et al., Science306(5705):2261-4. Epub Nov. 25, 2004). These committed mesenchymal cellscan then undergo a mesenchymal to epithelial transition to formendocrine cells. In another embodiment, non-islet exocrine cells may becultured and induced to adopt an endocrine phenotype (see Todorov etal., Pancreas 32(2):130-138, 2006). Both the non-islet exocrine cellsand the cells that are induced to adopt an endocrine phenotype can beidentified using the antibodies disclosed herein.

In one example, cultures can be initiated by labeling of live cells withduct-specific antibodies and isolation of positive cells using FACS orimmunomagnetic separation. Alternatively, depletion of cells expressingendocrine-specific antigens (and/or exocrine-specific antigens) could beemployed to enrich for exocrine and duct cells. An endocrine phenotypecan then be induced, and labeling with endocrine-specific antibodiesthen permits the non-destructive isolation of the subpopulation of cellsthat had begun to express and endocrine hormone, such as insulin.

Pancreatic endocrine cells (or a subset thereof) can further begenerated from liver cells (see Kojima et al., Nat. Med. (5):596-603.Epub 2003 Apr. 21, 2003). Pancreatic endocrine cells produced by thesemethods, or any other method known to one of skill in the art, can beisolated from other cell types using the antibodies disclosed herein.

In several examples, the isolated pancreatic endocrine cells or a subsetthereof are cultured in vitro, used in drug screening assays (such as toidentify agents of use in treating diabetes or pancreatic endocrine celltumors), or can be transplanted into a subject. Similarly, isolatedpancreatic exocrine cells or ductal cells can be cultured in vitro, andused in drug screening assays (such as to identify agents of use intreating pancreatic exocrine cell tumors or pancreatic adenocarcinomas,respectively). The methods include contacting the isolated pancreaticcells with an agent of interest and detecting the effect of the agent ofinterest on the pancreatic cells as compared to a control (such as acell contacted with a vehicle, or a cell not contacted with the agent).In several examples, the ability of the agent to cause death of thepancreatic cells, or to decrease cell division, is assessed. An increasein cell death, or a decrease in cell division, indicates the agent is ofuse to treat pancreatic cancer. In another example, the ability of theagent to alter secretion by the pancreatic cells is assessed.

In one embodiment, suspension of cells including pancreatic endocrinecells is produced, and one or more of the antibodies disclosed hereinis/are reacted with the cells in suspension. Methods of determining thepresence or absence of a cell surface marker are well known in the art.Typically, labeled antibodies specifically directed to the marker areused to identify the cell population.

Antibodies can be conjugated to other compounds including, but notlimited to, enzymes, paramagnetic beads, colloidal paramagnetic beads,haptens, fluorochromes, metal compounds, radioactive compounds or drugs.The enzymes that can be conjugated to the antibodies include, but arenot limited to, alkaline phosphatase, peroxidase, urease andB-galactosidase. The fluorochromes that can be conjugated to theantibodies include, but are not limited to, fluorescein isothiocyanate,tetramethylrhodamine isothiocyanate, phycoerythrin, allophycocyanins andTexas Red. For additional fluorochromes that can be conjugated toantibodies see Haugland, R. P., Molecular Probes: Handbook ofFluorescent Probes and Research Chemicals (1992-1994). The metalcompounds that can be conjugated to the antibodies include, but are notlimited to, ferritin, colloidal gold, and particularly, colloidalsuperparamagnetic beads. The haptens that can be conjugated to theantibodies include, but are not limited to, biotin, digoxigenin,oxazalone, and nitrophenol. The radioactive compounds that can beconjugated or incorporated into the antibodies are known to the art, andinclude but are not limited to technetium 99m (⁹⁹Tc), ¹²⁵ I and aminoacids comprising any radionuclides, including, but not limited to, ¹⁴C,³H and ³⁵S. Additional reagents are described above, and/or are known inthe art.

Fluorescence activated cell sorting (FACS) can be used to detect and/orseparate pancreatic endocrine cells (or a subset thereof) by contactingthe cells with an appropriately labeled antibody. FACS can also be usedto detect and/or separate pancreatic exocrine cells and/or pancreaticductal cells. In one embodiment, additional antibodies and FACS sortingcan further be used to produce isolated populations of pancreatic cells,such as but not limited to pancreatic endocrine cells. Combinations ofthe antibodies disclosed herein can be utilized in order to maximize theselectivity. An example would be the isolation of HIC1 2-B4 ^(positive)DHIC2 2-C12 ^(negative) HIC1 1-C10 ^(negative) DHIC3 5-H10 ^(negative)cells, which are positive for an endocrine marker and negative formarkers of alpha cell, exocrine cell, and duct cell identity. FACSemploys a plurality of color channels, low angle and obtuselight-scattering detection channels, and impedance channels, among othermore sophisticated levels of detection, to separate or sort cells. AnyFACS technique may be employed as long as it is not detrimental to theviability of the desired cells (for exemplary methods of FACS see U.S.Pat. No. 5,061,620, herein incorporated by reference).

However, other techniques of differing efficacy may be employed toisolate desired populations of cells. The separation techniques employedshould maximize the retention of viability of the fraction of the cellsto be collected. The particular technique employed will, of course,depend upon the efficiency of separation, cytotoxicity of the method,the ease and speed of separation, and what equipment and/or technicalskill is required. Separation procedures may include magneticseparation, using antibody-coated paramagnetic beads, affinitychromatography, cytotoxic agents, either joined to a monoclonal antibodyor used in conjunction with complement, and “panning”, which utilizes amonoclonal antibody attached to a solid matrix, or another convenienttechnique. Antibodies attached to paramagnetic beads and other solidmatrices, such as agarose beads, polystyrene beads, hollow fibermembranes and plastic petri dishes, allow for direct separation. Cellsthat are bound by the antibody can be removed from the cell suspensionby simply physically separating the solid support from the cellsuspension. The exact conditions and duration of incubation of the cellswith the solid phase-linked antibodies will depend upon several factorsspecific to the system employed. The selection of appropriateconditions, however, is well within the skill in the art.

The unbound cells then can be eluted or washed away with physiologicbuffer after sufficient time has been allowed for the cells expressing amarker of interest (such as an antigen that binds one or more of themonoclonal antibodies disclosed herein) to bind to the solid-phaselinked antibodies. The bound cells are then separated from the solidphase by any appropriate method, depending mainly upon the nature of thesolid phase and the antibody employed.

In one specific, non-limiting example, pancreatic endocrine cells or asubset thereof are positively selected by paramagnetic bead separation,wherein paramagnetic beads are coated with a monoclonal antibody thatspecifically binds pancreatic endocrine cells as disclosed herein. Thecells that are specifically bound by the monoclonal antibody are thenremoved from the paramagnetic beads. Release of the cells from theparamagnetic beads can be effected by methods known in the art. Thepurity of the isolated cells is then checked with a FACSCAN® flowcytometer (Becton Dickinson, San Jose, Calif.), for example, if sodesired. In one embodiment, paramagnetic bead separation is used tofirst separate a population of cells that do not express a marker, suchas pancreatic exocrine cell markers (for panning methods see Small etal., J Immunol Methods 3; 167(1-2):103-7, 1994, herein incorporated byreference.

Similar methods can be used to isolate pancreatic exocrine cells orpancreatic ductal cells, using paramagnetic beads coated with anantibody that specifically bind pancreatic exocrine cells or pancreaticductal cells, respectively. In some embodiments of these methods,paramagnetic bead separation is first used to separate cells that do notexpress pancreatic endocrine cell markers.

The antibodies disclosed herein can also be used to distinguish betweenuseful and contaminating cells in cultures intended to produce endocrinecells or a subset thereof, such as beta cells (or pancreatic ductalcells). A variety of different cell sources and/or culture environmentscan be used to produce beta cells in vitro (see above); the antibodiesdisclosed herein can be used to select cells of interest.

The antibodies disclosed herein can also be used to assess the efficacyof culture conditions. For example, the effectiveness of adding amountof a growth factor, nutrient or cytokine to an in vitro cultureconditions can be evaluated. In one example, an efficient yield ofendocrine cells indicates that the culture method can be used toefficiently produce endocrine cells or a subset thereof. For example,endocrine cells can be generated using a standard set of cultureconditions and a test set of culture conditions. The number of endocrinecells generated under the two sets of conditions can then be assessed.In one example, an increase in the number of endocrine cells generatedusing the test conditions indicate that these conditions are effectivefrom the production of pancreatic endocrine cells.

In one example, the antibodies disclosed herein are used to isolateand/or identify cells capable of initiating, maintaining, or enhancingepithelial-to-mesenchymal-transition (EMT) cultures. These cultures areestablished using human islet cell preparations, generally preparationsof cadaveric islets, and yield large numbers of cells with a detectablelevel of glucose-responsive insulin secretion (see Gershengorn et al.,supra, 2004). Cells capable of initiating, maintaining, or enhancingnon-islet derived cell cultures that yield insulin-secreting progeny canbe isolated and/or identified (see Todorov et al., Pancreas32(2):130-138, 2006). In another example, the antibodies disclosedherein are used to identify and/or select cells differentiated fromembryonic stem cells. Thus, the pre-culture input or post-culture outputof cells in such cultures can be optimized by viable selection using theantibodies disclosed herein.

The antibodies disclosed herein can also be used to detect pancreaticendocrine cells or a subset thereof, pancreatic exocrine cells, orpancreatic ductal cells in vivo. The antibodies disclosed herein canalso be used to detect pancreatic tumors in vivo. In one embodiment, amonoclonal antibody that binds the pancreatic cell population ofinterest is administered to the subject for a sufficient amount of timefor the antibody to localize to the pancreas (or tumor) in the subjectand to form an immune complex with the pancreatic cells (or tumor). Theimmune complex can then be detected. In one specific, non-limitingexample detection of an immune complex is performed byimmunoscintography. Other specific, non-limiting examples of immunecomplex detection include radiolocalization, radioimaging, magneticresonance imaging or fluorescence imaging. Once detected, in an ectopiclocation (as in a tumor) the test results can be used to assist in orguide surgical or other excision of a tumor. In one embodiment, theantibody is linked to an effector molecule. In one specific,non-limiting embodiment, the effector molecule is a detectable label.Specific, non-limiting examples of detectable labels include aradioactive isotope, an enzyme substrate, a co-factor, a ligand, achemiluminescent agent, a fluorescent agent, an electron dense reagent,a hapten, or an enzyme. In several examples, the antibody specificallybinds pancreatic endocrine cells, pancreatic exocrine cells, orpancreatic ductal cells.

The antibodies disclosed herein can be used to target a therapeuticagent to pancreatic cells. Treating pancreatic cells (as in a tumor) ina subject includes the administration of a monoclonal antibody complexedto an effector molecule, such as, but not limited to, a radioactiveisotope or other chemotherapeutic agent. In one embodiment, the antibodyis complexed to an effector molecule, such as a radioactive isotope, isadministered to a subject prior to surgery or treatment. In one example,an antibody that specifically binds pancreatic endocrine cells isadministered to a subject prior to treatment for diabetes or anendocrine cell tumor. In another embodiment, the antibody complexed toan effector molecule, such as a radioactive isotope, is administered toa subject following surgery or treatment. Thus, in another example, anantibody that specifically binds pancreatic endocrine cells isadministered to a subject following treatment for diabetes or a tumor.After a sufficient amount of time has elapsed to allow for theadministered radiolabeled antibody to localize to cells of interest, theislet cells or tumor is detected. In additional examples, an antibodythat specifically binds ductal cells can be administered to a subjectprior to, or following, treatment for a pancreatic adenocarcinoma. Thus,the effectiveness of the treatment can be assessed.

In one embodiment, an antibody that specifically binds a cell surfaceantigen on pancreatic cells and a secondary antibody are administered tothe subject for a sufficient amount of time for the monoclonal antibodyto form an immune complex on a pancreatic cell, (such as, but notlimited to, in the pancreatic islets of Langerhans and/or a tumor) andfor the secondary antibody to form an immune complex with the monoclonalantibody that binds pancreatic cells (such as, but not limited to, inthe pancreatic islets of Langerhans and/or a tumor). In one embodiment,the antibody that binds pancreatic cells is complexed with the secondaryantibody prior to their administration to the subject. In one specific,non-limiting embodiment, the secondary antibody is linked to adetectable label. In one embodiment, the immune complex, which includesa pancreatic cell, the monoclonal antibody that binds the pancreaticcell, and the secondary antibody linked to a detectable label, isdetected as described above. In several examples, the pancreatic cell isa pancreatic endocrine cell, a pancreatic exocrine cell, or a pancreaticductal cell.

Thus, in vivo imaging methods can also be utilized with the antibodiesdisclosed herein. These technologies include magnetic resonance imaging,positron emission tomography, and optical imaging. Advances in beta cellimaging using these technologies were recently reviewed (see Paty etal., Transplantation. 77:1133-1137, 2004, herein incorporated byreference). The methods include magnetic resonance imaging (such asusing an biotinylated antibody and avidin-iron oxide), positron emissiontomography (such as using an ¹¹¹indium-labeled monoclonal antibody), andoptical imaging (such as using luciferase or green fluorescent proteinlabeled antibodies). Recent data from the mouse indicates that betacells in the pancreas can be imaged using intact monoclonal antibodies,although the use of antibody fragments is also contemplated.

In one example, magnetic resonance imaging is utilized. In the settingof magnetic resonance imaging, contrast agent detection can be greatlyimpacted by magnetic resonance scanner field strength. Increased fieldstrengths provide improvements by orders of magnitude in the ability todetect contrast agents (Hu et al., Annu Rev Biomed Eng. 6:157-184, 2004;Wedeking et al., Magn. Reson. Imaging. 17:569-575, 1999). For example,the limit of detection of gadolinium at 2 tesla (T) is ˜30 μM. At 4T thelimit of detection is reduced to ˜1 μM. With newly available 7 to 12Tscanners one would expect to detect low (10-100) nM concentrations ofthis contrast agent. Similar sensitivity can also be identified usingcontrast agents such as iron oxide.

Kits are also provided herein. Kits for detecting pancreatic endocrinecells or a subset thereof will typically comprise an antibody thatspecifically binds a cell surface antigen on pancreatic endocrine cells,such as any of the antibodies described herein. Kits for detectingpancreatic exocrine cells will typically comprise an antibody thatspecifically binds a cell surface antigen on pancreatic exocrine cells,such as any of the antibodies described herein. Kits for detectingpancreatic ductal cells will typically comprise an antibody thatspecifically binds a cell surface antigen on pancreatic ductal cells,such as any of the antibodies described herein. More than one of theantibodies disclosed herein can be included in the kit. Thus, the kitcan include two or more of an antibody that specifically bindspancreatic endocrine cells, and/or an antibody that specifically bindspancreatic exocrine cells, and/or an antibody that specifically bindspancreatic ductal cells. In some embodiments, an antibody fragment, suchas an Fv fragment is included in the kit. In one example, such as for invivo uses, the antibody can be a scFv fragment. In a further embodiment,the antibody is labeled (for example, with a fluorescent, radioactive,or an enzymatic label).

In one embodiment, a kit includes instructional materials disclosingmeans of use of an antibody that specifically binds pancreatic cells.The instructional materials may be written, in an electronic form (suchas a computer diskette or compact disk) or may be visual (such as videofiles). The kits may also include additional components to facilitatethe particular application for which the kit is designed. Thus, forexample, the kit may additionally contain means of detecting a label(such as enzyme substrates for enzymatic labels, filter sets to detectfluorescent labels, appropriate secondary labels such as a secondaryantibody, or the like). The kits may additionally include buffers andother reagents routinely used for the practice of a particular method.Such kits and appropriate contents are well known to those of skill inthe art.

In one embodiment, the diagnostic kit comprises an immunoassay. Althoughthe details of the immunoassays may vary with the particular formatemployed, the method of detecting pancreatic cells (such as pancreaticendocrine cells or a subset thereof) in a biological sample generallyincludes the steps of contacting the biological sample with an antibodywhich specifically reacts, under immunologically reactive conditions, tothe pancreatic cells of interest (such as endocrine cells). The antibodyis allowed to specifically bind under immunologically reactiveconditions to form an immune complex, and the presence of the immunecomplex (bound antibody) is detected directly or indirectly.

Methods of determining the presence or absence of a cell surface markerare well known in the art. For example, the antibodies can be conjugatedto other compounds including, but not limited to, enzymes, paramagneticbeads, colloidal magnetic beads, haptens, fluorochromes, metalcompounds, radioactive compounds or drugs, as described herein. Theantibodies can also be utilized in immunoassays such as but not limitedto radioimmunoassays (RIAs), enzyme linked immunosorbant assays (ELISA),or immunohistochemical assays.

Pharmaceutical Compositions and Therapeutic Methods

Pharmaceutical compositions are disclosed herein that include anantibody that specifically binds pancreatic cells, such as pancreaticendocrine cells or a subset thereof, pancreatic exocrine cells, orpancreatic ductal cells (including a humanized form thereof or afunctional fragments thereof). These pharmaceutical compositions are foruse in methods of treatment and/or methods of detection, and can beformulated with an appropriate solid or liquid carrier, depending uponthe particular mode of administration chosen. In addition, a monoclonalantibody linked to an effector molecule (i.e., toxin, chemotherapeuticdrug, or detectable label) can be prepared in pharmaceuticalcompositions. Compositions including an antibody that specifically bindspancreatic endocrine cells or a subset thereof are of use, for example,for the treatment of pancreatic endocrine cell tumors, such as, but notlimited to, insulinomas, glucagonomas, or multiple endocrine neoplasmtype 1 (MEN-1). Compositions including an antibody that specificallybinds pancreatic exocrine cells are of use, for example, for thetreatment of pancreatic exocrine cell tumors. Compositions including anantibody that specifically binds pancreatic ductal cells are of use, forexample, for the treatment of pancreatic adenocarcinoma.

The pharmaceutically acceptable carriers and excipients useful in thisdisclosure, for either therapeutic or diagnostic methods, areconventional. For instance, parenteral formulations usually compriseinjectable fluids that are pharmaceutically and physiologicallyacceptable fluid vehicles such as water, physiological saline, otherbalanced salt solutions, aqueous dextrose, glycerol or the like.Excipients that can be included are, for instance, other proteins, suchas human serum albumin or plasma preparations. If desired, thepharmaceutical composition to be administered can also contain minoramounts of non-toxic auxiliary substances, such as wetting oremulsifying agents, preservatives, and pH buffering agents and the like,for example sodium acetate or sorbitan monolaurate.

The dosage form of the pharmaceutical composition will be determined bythe mode of administration chosen. For instance, in addition toinjectable fluids, topical, inhalation, oral and suppositoryformulations can be employed. Topical preparations can includeointments, sprays and the like. Inhalation preparations can be liquid(such as solutions or suspensions) and include mists, sprays and thelike. Oral formulations can be liquid (for example, syrups, solutions orsuspensions), or solid (such as powders, pills, tablets, or capsules).Suppository preparations can also be solid, gel, or in a suspensionform. For solid compositions, conventional non-toxic solid carriers caninclude pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. Actual methods of preparing such dosage forms are known, orwill be apparent, to those skilled in the art.

The pharmaceutical compositions that include a monoclonal antibody canbe formulated in unit dosage form suitable for individual administrationof precise dosages. In addition, the pharmaceutical compositions may beadministered in a single dose or as in a multiple dose schedule. Amultiple dose schedule is one in which a primary course of treatment maybe with more than one separate dose, for instance 1-10 doses, followedby other doses given at subsequent time intervals as needed to maintainor reinforce the action of the compositions. Treatment can involve dailyor multi-daily doses of compound(s) over a period of a few days tomonths, or even years. Thus, the dosage regime will also, at least inpart, be determined based on the particular needs of the subject to betreated and will be dependent upon the judgment of the administeringpractitioner. In one specific, non-limiting example, a unit dosage canbe about 0.1 to about 10 mg per patient per day. Dosages from about 0.1up to about 100 mg per patient per day may be used, particularly if theagent is administered to a secluded site and not into the circulatory orlymph system, such as into a body cavity, into a lumen of an organ, ordirectly into a tumor. In one embodiment, about 10 mCi of a radiolabeledmonoclonal antibody is administered to a subject. In other embodiments,about 15 mCi, about 20 mCi, about 50 mCi, about 75 mCi or about 100 mCiof a radiolabeled monoclonal antibody is administered to a subject. Theamount of active compound(s) administered will be dependent on thesubject being treated, the severity of the affliction, and the manner ofadministration, and is best left to the judgment of the prescribingclinician. Within these bounds, the formulation to be administered willcontain a quantity of the active component(s) in amounts effective toachieve the desired effect in the subject being treated.

The compounds of this disclosure can be administered to humans on whosetissues they are effective in various manners such as administrationinto the tumor. However, administration topically, orally,intravascularly such as intravenously, intramuscularly,intraperitoneally, intranasally, intradermally, intrathecally,subcutaneously, via inhalation or via suppository is of use with theantibodies disclosed herein. The particular mode of administration andthe dosage regimen will be selected by the attending clinician, takinginto account the particulars of the case (for example the subject, thedisease, the disease state involved, and whether the treatment isprophylactic).

In one embodiment, a therapeutically effective amount of an antibody isthe amount necessary to inhibit further growth of pancreatic endocrinecell tumor, a pancreatic exocrine cell tumor, or a pancreaticadenocarcinoma, or the amount that is effective at reducing a sign or asymptom of the tumor. In another embodiment, a therapeutically effectiveamount of an antibody is the amount sufficient to visualize pancreaticcells in a subject, such as but not limited to pancreatic endocrinecells (or a subset thereof) in the islets of Langerhans of the pancreas.It is advantageous for this dose to be administered in a human subjectwithout eliciting a human anti-mouse (HAMA) response in the subjectreceiving the treatment.

Controlled release parenteral formulations of a monoclonal antibody canbe made as implants, oily injections, or as particulate systems. For abroad overview of protein delivery systems (see Banga, A. J.,Therapeutic Peptides and Proteins: Formulation, Processing, and DeliverySystems, Technomic Publishing Company, Inc., Lancaster, Pa., 1995).Particulate systems include microspheres, microparticles, microcapsules,nanocapsules, nanospheres, and nanoparticles. Microcapsules contain thetherapeutic protein as a central core. In microspheres the therapeuticis dispersed throughout the particle. Particles, microspheres, andmicrocapsules smaller than about 1 μm are generally referred to asnanoparticles, nanospheres, and nanocapsules, respectively. Capillarieshave a diameter of approximately 5 μm so that only nanoparticles areadministered intravenously. Microparticles are typically around 100 μmin diameter and are administered subcutaneously or intramuscularly (seeKreuter, J., Colloidal Drug Delivery Systems, J. Kreuter, ed., MarcelDekker, Inc., New York, N.Y., pp. 219-342, 1994; Tice & Tabibi, Treatiseon Controlled Drug Delivery, A. Kydonieus, ed., Marcel Dekker, Inc. NewYork, N.Y., pp. 315-339, 1992).

Polymers can be used for ion-controlled release. Various degradable andnondegradable polymeric matrices for use in controlled drug delivery areknown in the art (Langer, R., Accounts Chem. Res. 26:537, 1993). Forexample, the block copolymer, polaxamer 407 exists as a viscous yetmobile liquid at low temperatures but forms a semisolid gel at bodytemperature. It has shown to be an effective vehicle for formulation andsustained delivery of recombinant interleukin-2 and urease (Johnston etal., Pharm. Res. 9:425, 1992; and Pec et al., J. Parent. Sci. Tech.44:58, 1990). Alternatively, hydroxyapatite has been used as amicrocarrier for controlled release of proteins (Ijntema et al., Int. J.Pharm. 112:215, 1994). In yet another aspect, liposomes are used forcontrolled release as well as drug targeting of the lipid-capsulateddrug (Betageri, et al., Liposome Drug Delivery Systems, TechnomicPublishing Co., Inc., Lancaster, Pa., 1993). Numerous additional systemsfor controlled delivery of therapeutic proteins are known (see, forexample, U.S. Pat. Nos. 5,055,303, 5,188,837, 4,235,871, 4,501,728,4,837,028 4,957,735 and 5,019,369, 5,055,303; 5,514,670; 5,413,797;5,268,164; 5,004,697; 4,902,505; 5,506,206, 5,271,961; 5,254,342 and5,534,496).

Site-specific administration of the disclosed compounds can be used, forinstance by applying the antibody a region of tissue from which a tumorhas been removed, or a region suspected of being prone to tumordevelopment. In some embodiments, sustained intra-tumoral (ornear-tumoral) release of the pharmaceutical preparation that includes atherapeutically effective amount of the antibody may be beneficial.

The present disclosure also includes therapeutic uses of monoclonalantibodies that are non-covalently or covalently linked to effectormolecules. In one specific embodiment, the monoclonal antibody iscovalently linked to an effector molecule that is toxic to a pancreatictumor. In one specific, non-limiting example, the effector molecule is acytotoxin. In other specific, non-limiting examples the effectormolecule is a radioactive isotope, a chemotherapeutic drug, abacterially-expressed toxin, a virally-expressed toxin, a venom protein,or a cytokine. Monoclonal antibodies covalently linked to an effectormolecule have a variety of uses. For example, an antibody linked to aradioactive isotope is of use in immunotherapy. An antibody covalentlylinked to a radioactive isotope is of use to localize a tumor inradioimmunoguided surgery, such that the tumor can be removed.

The present disclosure also includes combinations of a monoclonalantibody, with one or more other agents useful in the treatment oftumors. For example, the compounds of this disclosure can beadministered in combination with effective doses of immunostimulants,anti-cancer agents (such as chemotherapeutics), and/or vaccines. Theterm “administration in combination” or “co-administration” refers toboth concurrent and sequential administration of the active agents.

The disclosure is illustrated by the following non-limiting Examples.

EXAMPLES Example 1 Monoclonal Antibodies

For the generation of these antibodies, BALB/c mice were immunized withwhole cells (either intact islets or enzyme-dispersed islet cells), andtwo different immunization strategies have been used. The twoimmunization strategies include a “standard method” where animals areimmunized with desired target cells or a “subtractive method” designedto limit reactivity against undesirable antigens (Brooks et al., J. CellBiol. 122:1351-1359, 1993; Sleister et al., J Immunol Methods252:121-129, 2001; Sleister et al., J Immunol Methods. 261:213-220;Zijlstra et al., Biochem Biophys Res Commun. 303:733-744, 2003). Forsubtractive immunizations, mice were first given 2 million humanperipheral blood leukocytes (PBL) (as a source of undesirable humanantigens), followed by administration of cyclophosphamide on days 1 and2 after this initial immunization to ablate B-cells reactive with theundesirable PBL-associated antigens. The “subtracted” mice were thenimmunized 2 times with preparations of enriched islet cells obtained asislet equivalents from the Islet Cell Resource Centers. For standardimmunizations, mice were immunized 2-3 times with enriched islet cells.Four days after the final boosts (for both immunization strategies),splenocyes were fused with SP2/0 myeloma cells and plated inmethylcellulose media. 600-800 hybridoma clones (from each fusion) werepicked after 10-14 days of growth, and subcultured in 96-well plates.Supernatant from each clone was then used to stain acetone fixed frozenhuman pancreas sections in a primary screen, with afluorochrome-conjugated polyclonal anti-mouse immunoglobulin used todetect section-bound unlabeled primary antibodies. Stained sections wereanalyzed by fluorescence microscopy for novel antibodies reacting withdiscrete pancreatic cell subsets.

These fusions have yielded a large panel of antibodies that react withdifferent pancreatic cell subsets (Table 3). Monoclonal antibodies weredeveloped against pancreatic endocrine cells. The cell specificity ofeach monoclonal antibody was determined by real time polymerase chainreaction using FACS sorted cells. For the determination, cells weresorted using the specified monoclonal antibody, and the sorted cellswere evaluated for mRNA encoding insulin (β cells) or glucagon (αcells). Ratios of close to one indicate that message for both moleculeswas present, a ratios of much higher than one indicates selectiveexpression of insulin, and ratios of much less than one indicateselective expression of glucagon. Alternatively, cell specificity wasdetermined by two-color immunohistochemistry. Digital images obtainedfrom immunohistochemical studies using monoclonal antibodies that definecellular targets in pancreatic islets, are illustrated in FIG. 1. Eachpanel in this figure illustrates staining with antibody from anindependently derived hybridoma. Panels HIC0 4-F9, HIC1 2-B4, HIC1 4-G6,HIC1 5-F10 and HIC1 7-H10 illustrate staining of all islet cells(pan-islet). The panel labeled HIC0 3-05 illustrates staining with anantibody that binds to a subset of endocrine cells (including a subsetof (3 cells). Panels labeled DHIC2 2-B4 and DHIC2 2-C12 illustratestaining with antibodies that react with alpha cells, although DHIC22-B4 may react to a lesser extent with other pancreatic endocrine cells.Antibodies with duct-specific labeling are illustrated in panels labeledDHIC2 4A-10 and DHIC3 5-H10, and exocrine acinar labeling is shown inHIC1 1-C10. Sections of pancreas were incubated with primary antibodies,then with a Cy-3- or FITC-conjugated detection antibodies. Cellsreacting with primary antibody appear light against a dark background ofcells that do not react with the antibody.

TABLE 4 Monoclonal Antibodies Hybridoma/ Monoclonal ImmunoglobulinInsulin/ Cell Type Name Isotype Glucagon Specificity* HIC0 4-F9 IgG10.58 Pan-islet cells HIC1 2-B4 ND 1.23 Pan-islet cells HIC1 4-G6 ND 0.77Pan-islet cells HIC1 7-H10 IgG1 ND Pan-islet cells HIC0 3-C5 IgM 79Endocrine cell subset HIC1 5-F10 IgG1 14 Pan-islet cells DHIC2 2-B4 IgG1ND Alpha cells > other islet cells DHIC2 2-C12 IgM 0.001 Alpha cellsHIC1 1-C10 IgM NA Exocrine cells DHIC2 4-A10 IgG1 NA Duct cells DHIC35-H10 IgM NA Duct cells ND; not done. NA; not applicable. *Cell typespecificity defined insulin to glucagons ratio and/or by two colorimmunohistochemistry.

Example 2 Fluorescent Activated Cell Sorting (FACS)

For the in situ imaging of live beta cells, monoclonal antibodies mustreact with cell surface molecules on those cells. Flow cytometricanalyses of enzyme-dispersed islet cells have revealed that all of theantibodies react with cell surface molecules (see FIG. 2). For thisanalysis, intact islets obtained from the Islet Cell Resource Centerswere dispersed by treatment with trypsin (0.05% for 5-10 min at 37° C.).Enzyme-dispersed islet cells were then incubated with islet cellspecific antibodies, and bound primary antibody was assessed by flowcytometry using an APC-conjugated polyclonal anti-mouse immunoglobulinas a secondary reagent. The upper left panel illustrates levels ofsignal associated with the negative control. The other panels illustratestaining with the eleven antibodies introduced in FIG. 1.

Antibody reactivity with cell surface molecules is revealed by a rightshift in the fluorescent signal of a subset of the cells. Positive cellsare contained within the box on the right side of each panel, and thepercentage of cells identified as positive is indicated in each box. Theantibodies produced by the hybridomas HIC0 4-F9, HIC1 2-B4, HIC1 4-G6,HIC1 7-H10, HIC0 3-05, HIC1 5-F10, DHIC2 2-B4, or DHIC2 2-C12 all reactwith cell surface molecules on trypsin-dispersed pancreatic endocrinecells. The antibodies produced by hybridomas DHIC2 4A-10 and DHIC3 5-H10react with cell surface molecules on pancreatic duct-derived cells andantibodies produced by the hybridoma HIC1 1-C10 react with cell surfacemolecules on exocrine cells.

The INFLUX™ cell sorter is used on a routine basis to viably sort humanpancreatic islet cells, although other cell sorters can also beutilized. For sorting of individual islet cells, islets are dispersed bya brief exposure to trypsin (0.05% for 5-10 min at 37° C.).

Sorted and unsorted trypsin-dispersed islet cells were plated forgrowth. Trypsin-dispersed cells sorted using the INFLUX™ instrument havethe same proliferative potential as cells that had not been passed thruthe instrument.

In order to stain the human islets with the antibodies disclosed herein,the islets are dispersed into single cell suspensions by incubation withtrypsin (0.05% for 5-10 min at 37° C.), isolated cells are washed, andthen stained with candidate antibodies (using a fluorchrome-conjugatedsecondary anti-mouse immunoglobulin). Labeled cells are sorted intoislet cell populations that react with the antibody (positive cells) andpopulations that do not react with the antibody (negative cells). mRNAfrom these cell populations are subjected to real time polymerase chainreaction analyses using primers specific for insulin (as a beta cellspecific marker) and glucagon (as an alpha cell specific marker). Theinterpretation of these reactivity profiles would be that the candidateantibodies are beta cell specific, alpha cell specific, or labeled bothalpha and beta cells, respectively. These data are shown in Table 4.

Example 3 Additional Methods

An alternate method for determining cell specificity of antibodies is tostain cells with test antibodies as described above, then fix andpermeabilize the cells and stain for intracellular enzymes, insulin,glucagon, and amylase using polyclonal rabbit antibodies and analternate fluorochrome for enzyme detection.

A third strategy used to characterize the cellular specificities ofthese antibodies is two-color fluorescence microscopy. Pancreatic tissueis labelled with candidate mouse antibodies (with a Cy-3-conjugatedsecondary antibody as in the preliminary data section) and withpolyclonal rabbit antibodies directed against enzymes produced by knownislet cell subsets (with FITC). In some examples, antibodies thatspecifically bind glucagon or insulin are used. The anti-enzymeantibodies are obtained form commercial vendors as unconjugatedpolyclonal antibodies. These antibodies are biotinylated and detected ontissue sections using streptavidin conjugated to FITC. This strategydemonstrates that the antibodies co-label cells producing insulin only,glucagon only, or both enzymes. Thus, the antibodies disclosed hereincan readily be identified as beta cell specific, alpha cell specific, orlabeled both alpha and beta cells, respectively. Control slides stainedfor both insulin and glucagon are included in these analyses as acontrol, as antibodies to these enzymes should not co-label cells.Staining for both enzymes is accomplished with biotinylated antibodiesby using the following strategy: 1) incubate with the first biotinylatedantibody; 2) wash, then incubate with a streptavidin-fluorochromeconjugate (which will bind to the first antibody); 3) wash and blockwith free biotin (this step blocks available biotin binding sites onstreptavidin); 4) incubate with second biotinylated antibody; and 5)wash, then incubate with a distinct streptavidin-fluorochrome conjugate(which will bind the second antibody).

Example 4 Humanization and Production of an scFv

CDR amino acid sequences from monoclonal antibody HIC0 4-F9 (see TableI) are used for humanization and construction of recombinant scFv andscFv₂ fragments. A diagram illustrating the production of humanizedantibodies (and scFv) is shown in FIG. 3. The HIC0 4-F9 monoclonalantibody heavy-chain variable domain (Fv_(H)) has significant sequencehomology to the humanized Fab D3h44. Thus, the CDRs from HIC0 4-F9 areinserted into the D3h44 framework regions.

Protein-based and cell-based assays have been used extensively for thepurpose of evaluating engineered antibodies (reviewed by Qu et al,Methods. 36:84-95, 2005).

Competitive cell-based binding assays are developed to compare theantigen binding capabilities of engineered antibodies with those of theparental mouse monoclonal antibodies. For initial assays,trypsin-dispersed (0.05% for 5-10 minutes at 37° C.) human islets areused as a source of target cells. For these assays, unlabeled engineeredantibodies are used as a competitor of antigen binding by phycoerythrin(PE)-labeled parental antibodies. Briefly, dispersed islet cells areplated at 1×10⁵ cells/well in a 96-well plate (100 uL/well). A constantamount of PE-labeled parental antibody (10 nM) is mixed with varyingconcentrations of unlabeled parental or engineered antibodies (0.2-1,000nM) and added to each well (100 uL/well), with each experimentalcondition set up in triplicate. Plates are preblocked to prevent bindingof PE-conjugated antibody to the plate surface (phosphate bufferedsaline (PBS), 0.05% Tween 20, and 5% fetal calf serum (FCS) for 2 hoursat room temperature). After adding cells plus antibodies, the plates areincubated on ice with gentle mixing for 2 hr. Plates are thencentrifuged and washed five times to eliminate unbound PE-labeledantibody and evaluated for PE signal using a fluorescence plate reader.The fluorescence associated with cells is plotted versus theconcentration of unlabeled antibodies, yielding competitive inhibitioncurves. Successful engineering results in similar curves for theengineered and parental antibodies. Competitive radio-immunoassays canalso be used as an alternative for this determination.

Example 5 Identification of Protein Antigens and Development of Assays

Immunoaffinity column chromatography are used to isolate/enrich betacell associated target antigens (Nakache et al., Nature 337:179-181,1989). The enriched material is run on 1D SDS PAGE gels, and bandscorresponding to immunoreactive species (determined by Western Blot ofduplicate gels) is excised, subjected to tryptic digestion, and analyzedby nanoLC/MS/MS. Briefly, gel slices are washed to remove coomassiestain and then dehydrated by the addition of neat acetonitrile (ACN).Gel slices are treated with DTT and iodoacetamide to reduce and alkylatecystines. Prior to proteolysis, the gel slices are washed and driedagain. Proteolysis with trypsin is carried out overnight at 37° C., andpeptides are extracted from the gel slices by the addition of twoaliquots of 1% formic acid.

Protein identification and quantification is carried out using anApplied Biosystems Qstar XL. Briefly, 5 uL of peptides from the digestis injected onto a reverse phase trap column, washed thoroughly, andthen switched in-line with a 15 cm×75 uM analytical column packed withC18 reverse phase material. Peptides are eluted with an increasingorganic gradient (0-40% ACN) and introduced to the mass spectrometer viaan electrospray interface. Data dependent acquisition is used to selectprecursor ions and set collision energy for collisionally induceddissociation (CID) of the three most abundant ions derived from eachsurvey scan. Product ion spectra is used to obtain proteinidentification via database searching using the MASCOT™ (Matrix science)search engine.

Candidate antigen gene sequences are identified using GenBank and thosesequences are used to design primers for extracellular domain sequenceamplification. Type I proteins are expressed as human IgG1 Fc constructsand Type II proteins are expressed with an N-terminal Flag tag. Fusionproteins are detected and purified by their tags.

Extracellular domains of proteins of interest are amplified by PCR frombeta cell cDNA. Type I membrane proteins is cloned into a pCR-3mammalian expression vector (Invitrogen) modified to include thefollowing features: 1) a multiple cloning site for insertion of theextracellular domain of the protein; and 2) a cassette encoding thehinge, CH2 and CH3 domains of human IgG1 (GENBANK® accession numberX70421, as available on Apr. 17, 2006). Extracellular domains of Type IImembrane proteins are cloned into a pCR-3 vector modified to provide thefollowing elements: 1) the signal peptide of heamagglutinin to targetthe recombinant protein to the secretory pathway, 2) the eight aminoacid Flag sequence, 3) a linker sequence located between the Flag tagand the ligand and multiple cloning sites for the insertion of cDNA.

Beta cell cDNA are amplified with a high fidelity DNA polymerase, andthe PCR product is cloned into a pCR-blunt vector for sequencing. ThecDNA is recloned into the appropriate PCR-3 modified expression vectorsand used for either transient expression using 293T cells or stableexpression by HEK293 (ATCC CRL 1573). Fc-chimeric proteins are purifiedusing a protein A column (Pierce), while Flag-tag recombinant proteinare purified on an M2-agarose (Sigma) column.

Competitive protein-based binding assays are developed to compare theantigen binding capabilities of any humanized forms of the antibodiesdisclosed herein with those of the parental mouse monoclonal antibodies.For these assays wells of 96-well plates are coated with target antigen(target antigen at a concentration of 1 ug/mL in 50 uL of PBS). Theplates are then blocked to prevent binding of PE-conjugated antibody toplastic (PBS, 0.05% Tween 20, and 5% FCS for 2 hr at room temperature).After completion of the blocking step, a constant amount of PE-labeledparental antibody (10 nM) is mixed with varying concentrations ofunlabeled parental or engineered antibodies (0.2-1,000 nM) and added toeach well (100 uL/well), with each experimental condition set up intriplicate. The plates is incubated on ice with gentle mixing for 2hours, plates are washed 5 times to eliminate unbound PE-labeledantibody and evaluated for PE signal using a fluorescence plate reader.The fluorescence associated with the wells is plotted versus theconcentration of unlabeled antibodies, yielding competitive inhibitioncurves. Successful engineering results in similar curves for theengineered and parental antibodies.

Example 6 Imaging of Pancreatic Beta Cells in an Animal Model

The use of the monoclonal antibodies disclosed herein for imaging can beassessed using immunodeficient mice transplanted with human islets (seeMoore et al., Diabetes. 2001; 50:2231-2236). Islets are transplantedinto immunodeficient NOD-SCID mice (Fowler et al., Transplantation79:768-776, 2005). Briefly, human islets (500-2,000 per animal/pertissue site) are transplanted into these mice at ˜48 hours followingpancreas dissociation. Islets are delivered to two sites, the liver byinjection into the portal vein, and the kidney by injection between thecapsule and the renal parenchyma. At 4 and 8 weeks post islettransplant, time points when transplanted islets are well vascularized,candidate beta cell specific imaging reagents are injected intravenously(iv) into transplant recipient animals. Animals receive graded doses (10ug, 1 ug, 0.1 ug, or 0.01 ug) of imaging reagents in a volume of 50 uL,and at 12 and 24 hr after reagent injection, animals are euthanized andliver and kidney is recovered and evaluated for presence of the imagingreagents and specific association of those reagents with transplantedbeta cells. The injected imaging reagents are FITC-conjugated,PE-conjugated, and biotin-conjugated. Controls include: Parentalantibodies; antibodies with specificities unrelated to islets; andengineered antibodies that lack specificity for human beta cells.

Tissues recovered from mice receiving candidate imaging reagents orcontrol reagents are processed for tissue section analyses and flowcytometric analyses. High resolution studies of beta cell targeting byinjected imaging reagents are conducted by evaluating sections fromfrozen tissue. For these analyses, thin (5 um) sections of frozen tissueare fixed in acetone, stained as required, and evaluated by fluorescencemicroscopy. With reagents labeled with FITC or PE, no additionalstaining is required for detection. For sections containing reagentslabeled with biotin, incubation with streptavidin-FITC, —PE or alternatestreptavidin conjugate is required for detection. Biotinylated reagentsare used to allow for greater flexibility in designing detection schemesand because biotin-streptavidin (or biotin-avidin) detection systemsallow for substantial signal amplification. Although islets are easy todetect in H&E stained sections, sections can also be stained withantibodies to human insulin and to human HLA Class I molecules toprecisely localize human beta cells and human islets, respectively.These antibodies are labeled with fluorochromes distinct from those usedto identify candidate imaging reagents, to allow detection of beta cellimaging reagent (the engineered antibodies), beta cells (insulinexpressing) and islet cells (HLA Class I expressing) within the sametissue section.

Islets transplanted under the kidney capsule can be readily dissociated,by micro-dissection, from renal parenchyma. Following recovery, theseislets are enzymatically dispersed using trypsin (0.05% for 5-10 minutesat 37° C.) and evaluated by flow cytometry. Detection strategies forengineered imaging reagents, beta cells, and islet cells are similar tothose described above. However, cells are permeablized to allowdetection of insulin or internalized biotinylated imaging antibody.

Example 7 General Methods

Larger Scale Antibody Preparations. For larger scale antibodypreparations, hybridomas are grown in serum-free media (Hybridoma-SFM;Gibco). Hybridoma growth in this media generally yields antibody at aconcentration of 5-20 mg/L. Following exhaustion of cultures, antibodyis recovered/purified from culture supernatant using immobilized proteinG (Pierce; as per manufacturer's instructions;http://www.piercenet.com/). Protein G works well for the isolation ofmost mouse immunoglobulin isotypes. Concentration/size exclusionchromatography is used for mouse IgM.

Immunoaffinity Isolation of Beta Cell Antigens. Beta cell antigens areimmunoaffinity purified from triton X-114 lysates of pancreatic isletsusing AminoLink affinity columns obtained from Pierce and used as permanufacturer's instructions (available on the internet, see the Piercewebsite). Briefly, purified monoclonal antibodies targeting cell surfacemolecules on pancreatic beta cells are covalently linked to AMINO LINK™coupling gel columns. Then remaining active sites on the columns areblocked and the columns washed. Columns are then equilibrated andantigen-containing sample is applied. Columns are then incubated toallow maximal binding of beta cell antigen, washed to remove undesirablematerials, and treated with elution buffer to promote release of boundantigen. Antigen containing solution is then neutralized, the buffer isexchanged to an appropriate working or storage buffer.

Antibody/Protein Labeling. To facilitate studies using more than oneantibody or where directly conjugated antibody is desirable, purifiedantibodies are conjugated to biotin and/or different fluorochromes(R-Phycoerythrin and NHS-Fluorescein). These fluorochromes have distinctemission spectra and can therefore be distinguished when used together.NHS-LC-biotin is used for antibody biotinylation and offers thepotential for signal amplification and/or the use of additionalfluorochromes (as avidin or streptavidin conjugates). The fluorochromesand biotin are obtained from Pierce, and are used as per manufacturer'sinstructions (available on the internet at the Pierce website). By usingantibodies labeled with different fluorchromes, tissue sections, celllines, enzymatically dispersed cells, or intact islets can be stainedwith two or more antibodies at the same time.

Example 8 Method for the Isolation of Pancreatic Endocrine Cells

There is a need to provide a means for the isolation of large numbers ofpancreatic endocrine cells or pancreatic endocrine-like cells, derivedfrom donor tissue or from ex vivo culture. Paramagnetic bead separation,using the monoclonal antibodies disclosed here, can be employed toseparate large numbers of pancreatic endocrine cells. For this analysis,intact islets obtained from a pancreas, such as a human cadavericpancreas, or culture derived islet-like cells are dispersed by treatmentwith trypsin (0.05% for 5-10 minutes at 37° C.). Enzyme-dispersed cellsare then incubated with a combination of monoclonal antibodies. Cellswith associated monoclonal antibodies are incubated with anti-mouseimmunoglobulin-coated paramagnetic beads. The cells targeted by themonoclonal antibodies are bound to the paramagnetic beads and areseparated from the cells that are not bound by the beads using a magnet.Cells not attracted to the magnet are removed by washing the cells whileexposed to the magnet. Cells attracted to the magnet are retained in themagnetic field. Upon removal from the magnetic field, paramagnetic beadassociated cells are recovered.

In one protocol, to achieve maximum purity, rounds of negative selection(by collection of cells which were not retained on the column afterlabeling with markers of duct or exocrine identity) are followed by around of positive selection (by collection of cells which were retainedon the column after labeling with markers of endocrine identity). Anexample is the depletion of cells labeled by DHIC2 2-C12, HIC1 1-C10 orDHIC3 5-H10 (markers of alpha cell, exocrine cell, and duct cellidentity) and then positive selection for cells labeled with HIC1 2-B4(pan-endocrine). The result is a pure population of beta cells.

Alternatives to the indirect separation strategy detailed above includeuse of paramagnetic beads coated with the pancreatic endocrine specificmonoclonal antibodies described herein, or avidin- orstreptavidin-coated paramagnetic beads. When avidin- orstreptavidin-coated beads are used, the pancreatic endocrine specificmonoclonal antibodies need to be biotinylated. Thus, the antibodiesdisclosed herein can be used to isolate pancreatic endocrine cells.

Example 9 Method for the Ex Vivo Generation of Pancreatic EndocrineCells

The antibodies disclosed herein can also be used to distinguish betweenuseful and contaminating cells in cultures intended to produce beta-likeor endocrine-like cells. A variety of different cell sources and/orculture environments may provide beta-like or endocrine-like activities,and these antibodies can be used to select for these characteristics.

For example, cells of interest can be produced in vitro by culturinghuman cadaveric islets; these cultures areepithelial-to-mesenchymal-transition (EMT) cultures. These cultures areestablished using human islet cell preparations, and yield large numbersof cells with a detectable level of glucose-responsive insulin secretion(see Gershengorn et al., supra, 2004). The islets and/or cells can beisolated from a pancreas obtained from a cadaver using the antibodiesdisclosed herein. In another instance, non-islet exocrine cells may becultured and induced to adopt an endocrine phenotype (see Todorov etal., Pancreas 32(2):130-138, 2006). The appropriate input populationsfor these cultures can be determined and obtained by positive ornegative labeling of live cells using the antibodies described here. Anexample of the differential potential of pancreatic subpopulations thatcan be discriminated using these antibodies is illustrated in FIG. 3 andTable 5. Isolation of HIC1 7-H10^(hi/+) cells from a mesenchymal phaseculture derived from human islets prior to the mesenchymal-to-epithelialtransition resulted in a more complete transition (as assessedmorphologically) and an enhanced level of insulin expression. Althoughcell lines established with islets from different patients or bydifferent methodologies may exhibit distinct characteristics (forexample, in some cases the HIC1 7-H10^(low/−) cells could be the moreproductive population), these antibodies can be used to obtain eitherpopulation. Regardless of the tissue of origin, the output cells fromthese cultures can be selected using the antibodies disclosed herein.Thus, viable pancreatic endocrine cells are purified using theantibodies disclosed herein.

TABLE 5 Cell culture to generate insulin-expressing cells SortedIslet-like Insulin mRNA subpopulation Mesenchymal cluster expression inICAs from EMET phase fold formation (relative to culture expansion (1w)efficiency* unsorted) HIC1 7-H10^(hi/+) 58x High 251x HIC1 7-H10^(low/−)23x None/Very low N/A N/A: Not applicable *Judged by the proportion ofcells which formed spheroid cultures associated with epithelialtransition

It is estimated that a diabetic subject will need at least about 10,000,or between 5,000 and 30,000 islets per kilogram body weight (or aequivalent number of pancreatic endocrine cells) per transplantation tohave a substantial beneficial effect from the transplantation. In onespecific, non-limiting example the cells are administered bysub-cutaneous injection, or by implantation under the kidney capsule,through the portal vein of the liver, or into the spleen. If, based onthe method of administration, cell survival after transplantation ingeneral is low (5-10%) additional islets, such as up to 100,000 isletsper kilogram body weight, are transplanted.

Transplantation can be achieved by injection. Injections can generallybe made with a sterilized syringe having an 18-23 gauge needle. Forexample, a pancreatic endocrine cell suspension or islets istransplanted using a needle not bigger than 1 mm in diameter. The cellsare administered by subcutaneous injection, intra-peritoneal injection,injection under the kidney capsule, injection through the portal vein,and/or injection into the spleen. The cells can be encapsulated prior toadministration, such as by co-incubation with a biocompatible matrixknown in the art. A variety of encapsulation technologies have beendeveloped (e.g., Lacy et al., Science 254:1782-84, 1991; Sullivan etal., Science 252:7180712, 1991; WO 91/10470; WO 91/10425; U.S. Pat. No.5,837,234; U.S. Pat. No. 5,011,472; U.S. Pat. No. 4,892,538, each hereinincorporated by reference).

The cells can be implanted using an alginate-polylysine encapsulationtechnique (O'Shea and Sun, Diabetes 35:943-946, 1986; Frischy et al.,Diabetes 40:37, 1991). In this method, the cells are suspended in 1.3%sodium alginate and encapsulated by extrusion of drops of thecell/alginate suspension through a syringe into CaCl₂. After severalwashing steps, the droplets are suspended in polylysine and rewashed.The alginate within the capsules is then reliquified by suspension in 1mM EGTA and then rewashed with Krebs balanced salt buffer. Each capsuleis designed to contain several hundred cells and have a diameter ofapproximately 1 mm. Capsules containing cells are implantedintraperitoneally and blood samples taken daily for monitoring of bloodglucose and insulin.

Example 10 Use of Monoclonal Antibodies to Isolate Cells for Use in aBioartificial Pancreas

There is a need to provide a biocompatible and implantable devicecontaining islets of Langerhans, or the insulin producing β cells, thatcan supply the hormone insulin for the purpose of controlling bloodglucose levels in people with diabetes mellitus requiring insulin.Insufficient regulation of blood glucose levels in people with diabeteshas been associated with the development of long-term health problemssuch as kidney disease, blindness, coronary artery disease, stroke, andgangrene resulting in amputation. Therefore, there is a need to replaceconventional insulin injections with a device that can provide moreprecise control of blood glucose levels.

Many modalities are currently available to replace the impairedpancreatic beta cell function in diabetes mellitus patients. Theelectromechanical modality utilizes insulin delivery systems thatrelease insulin in response to blood glucose levels that arecontinuously measured via a glucose sensor. Difficulties with thesensors led to the development of programmed insulin delivery via acontinuous perfusion pump. This approach however also falls short of thein vivo regulation, i.e. the regulation of insulin secretion by glucoseand its modulation by several hormonal and neuronal factors.

To overcome these problems, bioartificial pancreases have beendeveloped. These systems separate the transplanted tissue from thediabetic recipient by an artificial barrier, which diminishes immunerejection, yet allows the transfer of the glycemic signal from the bloodto the islet cells and the transfer of the pancreatic hormones from theislet cells to the blood. An artificial pancreas accomplishes this byhaving a selectively permeable barrier, which is permeable to glucoseand insulin, but not to immunoglobulins and immunocytes. Artificialpancreas devices work based on the transfer through the membrane of aglycemic signal from blood to the pancreatic endocrine cells, andinsulin from the pancreatic endocrine cells to the recipient. In oneembodiment, the pancreatic endocrine cells are in the form of islets.

In general, the transfer of a substance from one compartment to theother across a membrane can be achieved either by diffusion, dialysis,or by convection, ultrafiltration or a combination of these methods.Artificial pancreases are generally divided among those that utilizediffusion mechanisms, those that utilize convection mechanisms, or thosethat utilize a combination of both mechanisms. Diffusion represents thetransfer of the substance itself without transfer of the solvent.Convection, in contrast, involves the transfer of the solvent and anymolecules dissolved therein as long as they are smaller than the poresof the membrane.

Suitable devices for use with pancreatic endocrine cells as anartificial pancreas are well known in the art. Specific, non-limitingexamples devices of use are disclosed in U.S. Pat. No. 5,741,334; U.S.Pat. No. 5,702,444; U.S. Pat. No. 5,855,616; U.S. Pat. No. 5,913,998;U.S. Pat. Nos. 6,023,009; and 6,165,225, all of which are incorporatedby reference herein.

Thus, the antibodies disclosed herein can be used to isolate pancreaticendocrine cells. These cells are then included in a device as abioartificial pancreas, and the bioartificial pancreas is then implantedinto a subject. The implantation of the bioartificial pancreas resultsin the treatment of a disorder. In embodiment, the implantation of thebioartificial pancreas results in the treatment of diabetes.

Example 11 Antibodies that Specifically Bind Pancreatic Ductal Cells

Multiple monoclonal antibodies that meet the criteria of staining normalduct epithelium and pancreatic adenocarcinoma cells have beenidentified. FIG. 5 illustrates two independently derived monoclonalantibodies with this specificity profile. Reactivity with normal ductepithelium is shown in panels A and C, while extensive reactivity withpancreatic adenocarcinoma cells is shown in panels B and D. For theresults illustrated in this figure, supernatants from hybridomas weretested on acetone or methanol fixed frozen human pancreas sections(either normal pancreas or pancreatic adenocarcinoma), and aperoxidase-conjugated polyclonal secondary antibody was used to detectsection-bound unlabeled primary antibody. Addition of the chromogen3,3′-diaminobenzidine tetrahydrochloride (DAB) allowed visualization ofbound antibody (dark staining) against a light background of cells thatwere not bound by antibody. Stained sections were analyzed by lightmicroscopy.

In addition to characterization on specimens of normal and neoplasticpancreas, the two antibodies illustrated in FIGS. 1 and 5 have beenevaluated on a variety of additional tissues (summarized in Table 6).

TABLE 6 Cell and Tissue Distribution of Duct Reactive Antibodies.Antibody Designation Tissue DHIC2 4-A10 DHIC3 5-H10 Normal Pancreas + +(Duct epithelium) (Duct epithelium) Liver + + (Duct epithelium) (Ductepithelium) Breast − − Kidney − − Ovary − − Stomach ND* − NeoplasticPancreatic adenocarcinoma + + Liver cholangiocarcinoma + + Ovariancholangiocarcinoma ND + Liver hepatocellular − − carcinoma Breastadenocarcinoma − − Stomach adenocarcinoma + + Lung adenocarcinoma + +Kidney carcinoma − − Ovarian Endocarcinoma ND − GIST − − Leiomyosarcoma− − *ND, not determined

The results with monoclonal antibodies DHIC2 4-A10 and DHICH3 5-H10suggest that duct cells in diverse tissues exhibit tissue-selectivemarker expression profiles (for example, these two antibodies react withmarkers on pancreatic and liver duct cells, but they do not react withduct cells in mammary gland). Staining of adenocarcinomas derived fromdifferent tissues indicates that adenocarcinomas also exhibit distinctmarker profiles (for example, these antibodies react with pancreaticadenocarcinoma and do not react with breast adenocarcinoma or liverhepatocellular carcinoma). The results suggest that these antibodies areof use in the diagnosis of pancreatic adenocarcinoma.

The cell surface reactivity profiles of the two duct-reactive antibodiesis illustrated in FIG. 2. For this analysis, pancreatic cells (enrichedfor islet content—see figure legend) obtained from the Islet CellResource Centers were dispersed by treatment with trypsin (0.05% for 5min). Enzyme-dispersed cells were then incubated with candidateantibodies and bound primary antibodies were assessed by flow cytometryusing APC- or PE-conjugated polyclonal immunoglobulin as a secondaryreagent. The left panels illustrate levels of signal associated with thenegative controls, and right panels illustrate cell surface stainingwith duct reactive antibodies. These data demonstrate cell surfaceexpression of antigens recognized by these two antibodies on roughly 10%of cells within the pancreatic cell preparation.

Pancreatic ductal adenocarcinoma is the most lethal of human solidcancers. Although the incidence of this cancer is relatively low atapproximately 32,000 newly diagnosed cases per year in the USA (Jemal,Cancer statistics, CA Cancer J. Clin. 2005; 55:10-30, 2005), the currentfive-year survival rate following initial diagnosis is 1-5% (see Wray etal, Gastroenterology 128:1626-1641, 2005). As a consequence of the highmortality rate for patients with pancreatic adenocarcinoma, this canceris the fourth leading cause of cancer-related deaths for men and thefifth leading cause of cancer-related deaths for women in the USA (seeWingo et al., Cancer 97:3133-3275, 2003). Despite continuing substantiveefforts to alter the disease course in patients with pancreaticadenocarcinoma, conventional therapies including radiation and/orchemotherapy have had little impact on this aggressive disease (Hawes etal., Am J. Gastroenterol. 95:17-31, 2000). At present, tumor resectionduring early stage disease is the only potentially curative option forthese patients (Sener et al., J Am Coll Surg. 189:1-7, 1999).

A primary reason for the low survival rate for patients with pancreaticadenocarcinoma is the inability to diagnose this cancer during earlystage disease. At present, most patients are diagnosed with advanceddisease, with only 15% of newly diagnosed patients presenting withlocalized operable cancer (Li et al, Lancet 363:1049-1057, 2004). As theinability to detect this cancer at early stages is viewed as asignificant barrier to the effective treatment of these patients, thereis substantial interest from the research community, in defining newmarkers of early disease. The survival rate for patients with pancreaticadenocarcinomas is also negatively impacted by the lack of effectivetherapies. Long-term survival of patients undergoing resection oflocalized operable tumors is approximately 20% (see for example, Schmidtet al., Arch Surg. 139:718-725 and 725-717, 2004). Thus, the developmentof novel therapies is also viewed as critically important for successfuldisease management.

Most patients diagnosed with pancreatic adenocarcinoma have a lifeexpectancy of months rather than years. The poor prognosis for thesepatients is due to 1) the inability to detect early stage disease, 2)the metastasis of these tumors to distant sites early during the courseof this disease, and 3) the resistance of the disease to conventionalchemotherapy and/or radiation therapy. For patients with tumors locatedin the head and body of the pancreas, symptoms of disease are associatedwith compression of the bile duct, the pancreatic duct, the mesentericand celiac nerves, and the duodenum (Evans D B A J, Willett C G. Cancerof the pancreas. Philadelphia: Lippincott, Williams & Wilkins; 2001);and these tumors may or may not cause the patient pain. For tumorslocated in the tail of the pancreas, patients may have pain on the leftside of the abdomen, but pain is generally associated with late stagedisease. Thus, patients with pancreatic adenocarcinoma do not generallyseek treatment during early stage disease.

As patient survival clearly depends on early detection of this disease,there is tremendous interest in the identification of markers that canbe used for disease detection and diagnosis. Candidate serum markers ofdisease include: the sialylated Lewis^(a) blood group antigen CA19-9(see Gupta et al., Cancer 56:277-283, 1985), macrophage inhibitorycytokine-1 (MIC-1; also known as placental TGF-beta (Moore et al., JClin Endocrinol Metab. 85:4781-4788, 2000), prostate-derived factor(Karan et al., Biochem Biophys Res Commun. 305:598-604, 2003), andgrowth/differentiation factor 15 (Koniaris, J Gastrointest Surg.7:901-905, 2003); and osteopontin (Kolb et all, Cancer Biol Ther.4:740-746, 2005). Of these markers, CA19-9 has been the most widelystudied. Unfortunately, the results from those studies do not supportthe use of this marker in disease detection and diagnosis, particularlyin the diagnosis of early disease, where a high frequency of patientstest negatively for CA19-9 (Sawabu et al., Pancreas 28:263-267, 2004).In addition to the false-negative results associated with detection ofearly disease, false-positive results are obtained at high frequency inpatients with acute cholangitis (Albert et al., Dig Dis Sci.33:1223-1225, 19888) and chronic pancreatitis (Furuya et al., Br JCancer 73:372-376, 1996). Current data suggest that MIC-1 may be abetter marker of pancreatic adenocarcinoma than CA19-9 (Koopmann et al.,Clin Cancer Res. 10:2386-2392, 2004). However, MIC-1 is also present athigh frequency in patients with pancreatitis (Koopmann et al., supra).Osteopontin has also not allowed robust detection of disease (Kolb etal., Cancer Biol Ther. 4:740-746, 2005) Thus, there is currently noserum marker than can be used to accurately detect and diagnose earlystage pancreatic adenocarcinoma.

One treatment for patients with advanced pancreatic adenocarcinoma isthe chemotherapeutic agent gemcitabine (Burris et al., J ClinOnco1.15:2403-2413, 1997). This drug is a nucleoside analogue thatinhibits cell division, by interfering with DNA and RNA synthesis, andyields a modest improvement in survival and clinical benefit (Burris etal, supra). The drug combination of gemcitabine administered incombination with erlotinib (Tarceva) may have activity in advanceddisease. Tarceva is a small molecule that targets the epidermal growthfactor receptor 1 (EGFR) pathway and inhibits tyrosine kinase signaling(Ciardiello et al, Clin Cancer Res. 7:2958-297, 2001). In a phase IIImulti-center study involving 569 patients, patients treated with Tarcevaplus gemcitabine exhibited an improvement in overall survival of 23%when they were compared to patients receiving gemcitabine plus placebo(Moore et al., ASCO Annual Meeting, Abstract #1; 2005), and after oneyear of treatment, 23% of patients receiving Tarceva plus gemcitabinewere alive while only 17% of patients receiving gemcitabine plus placebowere alive. Thus, this combination of agents appears to offer anincremental improvement in outcomes in patients with advanced disease.

An alternate strategy to overcome pancreatic adenocarcinoma is by usingmonoclonal antibodies. One such monoclonal antibody, cetuximab, ahumanized monoclonal antibody directed against the EGFR has completedphase II trials and shows promise in patients with EGFR positivepancreatic adenocarcinoma. This antibody blocks activation of the EGFRtyrosine kinase, and effectively blocks the mitogenic signal deliveredby ligand binding of this receptor. In the phase II trial, patients weregiven cetuximab and gemcitabine, and at 1 year, patients who receivedthe combination therapy had an overall survival rate of 31.7% and aprogression fee survival rate of 12% (Xiong et al., J Clin Oncol.22:2610-2616, 2004). This compares favorably with an overall survivalrate of 18% and a progression fee survival rate of 9% from a previousphase III trial using gemcitabine alone (Burris et al., J Clin Oncol.15:2403-2413, 1997). A second monoclonal antibody, avastin, is alsobeing investigated in patients with pancreatic adenocarcinoma. Thismonoclonal antibody, directed against the VEGF receptor, yielded a 1year survival rate of 29% when administered with gemcitabine (Kindler etal, Bevacizumab (B) plus gemcitabine (G) in patient (pts) with advancedpancreatic cancer (PC): Updated results of a multi-center phase IItrial. Annual Meeting Proceedings (Post-Meeting Edition) Vol 22, No 14S(July 15 Supplement), 2004: 4009, 2004). A phase III trial has beeninitiated to validate results of the phase II study. The monoclonalantibodies disclosed herein can be used with any of these treatmentmodalities described above.

Example 12 Additional Studies

Serum and pancreatic duct fluid specimens are evaluated from patientswith pancreatic adenocarcinoma for the presence of tumor antigen. Forthis series of tests, serum and duct fluid specimens are evaluated from20-30 pancreatic cancer patients using Western blot analyses. Antibodyreactivity with a minimum of 10 serum specimens from normal donors(negative controls) is also assessed. Duct fluid from organ donors (fromnormal donors and from donors with pancreatitis) can also be assessed.The fluid is a negative duct fluid control. Blood samples (10 mL) forpreparation of serum is obtained and serum is aliquoted and stored at−80° C. until used. Pancreatic ductal fluid is obtained from cancerpatients during pancreatic resection for cancer. This is mixed with acomplete mini-protease inhibitor (Roche), aliquoted and stored at −80°C. until used.

There is a growing body of literature on the detection of cancer cellsin peripheral blood (see for example, Hayes et al., Clin Cancer Res.12:4218-24, 2006). The detection of circulating cancer cells can be usedfor the assessment of tumor cell tumorigenic potential or theresponsiveness of particular cancers to candidate therapeutic agents. Asthe antibodies described herein have been shown to react with cellsurface molecules, they are ideally suited for assessment and sorting ofcirculating live tumor cells from patients with pancreaticadenocarcinoma. Circulating cancer cells are present at low frequencies,ranging from 1 in 10³ to 1 in 10⁷ white blood cells. To determinewhether pancreatic adenocarcinoma cells are present in peripheral blood,flow cytometry and RT-PCR can be utilized. The detection, quantitation,and isolation of rare cells from peripheral blood requires a multi-steppreparative process. Briefly, patient blood is collected in tubescontaining an anti-coagulant (lithium heparin or sodium citrate). Redblood cells are lysed using an red blood cell lysis buffer(eBiosciences). Ficoll is not used, as it may result in loss of rarecells in the red blood cell pellet. Cells remaining following red bloodcell lysis are stained with a Fluorescein Isothyocyanate(FITC)-conjugated antibody directed against CD45 (a marker expressed onall hematopoietic cells and not on adenocarcinoma cells), withR-Phycoerythrin (PE)-conjugated antibodies directed againstadenocarcinoma cells (to detect cancer cells), and with propidium iodide(PI; a DNA-binding dye that reacts with dead cells). When the cells areanalyzed or sorted, most cells are excluded from the analysis as theyare PI positive (dead) or FITC positive (hematopoietic cells). Cellsthat do not stain with PI or FITC are assessed for the presence of PE.Cells that are PI negative, FITC negative, and PE positive are sorted ascandidate circulating cancer cells. A cancer origin of the cells isconfirmed using RT-PCR, where sorted cells are evaluated for thepresence of cytokeratin 19 mRNA. Controls include isotype controlantibodies and peripheral blood from normal donors.

Example 13 Protein Identification by Proteomic Analyses and AdditionalMethods

Immunoaffinity column chromatography are used to isolate/enrich thetarget protein (Nakache et al, Nature 337:179-181, 1989). Briefly,monoclonal antibodies at concentrations of 1-5 mg/mL are covalentlycoupled to an agarose matrix (AminoLink gel; Pierce; as per themanufacturer's instructions). This material is used as the affinitymatrix for column-based immunoaffinity purificiation of antigen. Forantigen purification, cell or tissue lysates containing target antigensare loaded onto these columns at neutral pH, columns are washed toeliminate contaminating proteins, and target antigens are eluted byadjusting the buffer pH to ˜2.8. Elution fractions containing targetantigen are identified by dot blot analyses.

Column enriched protein antigens are run on 1D SDS PAGE gels, and bandscorresponding to immunoreactive species (determined by Western Blot ofduplicate gels) are excised, subjected to tryptic digestion, andanalyzed by nanoLC/MS/MS. Briefly, gel slices are washed to removecoomassie stain and then dehydrated by the addition of neat acetonitrile(ACN). Gel slices are treated with DTT and iodoacetamide to reduce andalkylate cystines, and prior to proteolysis, the gel slices are washedand dried again. Proteolysis with trypsin is carried out overnight at37° C. Peptides are extracted from the gel slices by the addition of twoaliquots of 1% formic acid.

Protein identification and quantification is carried out using anApplied Biosystems Qstar XL. Briefly, 5 ul of peptides from the digestare injected onto a reverse phase trap column, washed thoroughly, andthen switched in-line with a 15 cm×75 uM analytical column packed withC18 reverse phase material. Peptides are eluted with an increasingorganic gradient (0-40% ACN) and introduced to the mass spectrometer viaan electrospray interface. Data dependent acquisition is used to selectprecursor ions and set collision energy for collisionally induceddissociation (CID) of the three most abundant ions derived from eachsurvey scan. Production spectra is used to obtain protein identificationvia database searching using the MASCOT™ (Matrix science) search engine.

Large scale production of antibodies are accomplished by growinghybridoms in the CELLLINE™ System (BD Biosciences) using serum-free lowprotein media (Hybridoma-SFM; Gibco). These cultures generally yieldsantibody at concentrations of 2-20 milligrams/mL, with contaminatingproteins present in the low microgram/mL range (such that the antibodiesare usually >98% pure).

It will be apparent that the precise details of the methods orcompositions described may be varied or modified without departing fromthe spirit of the described invention. We claim all such modificationsand variations that fall within the scope and spirit of the claimsbelow.

1. An isolated monoclonal antibody comprising a heavy chain comprisingan H-CDR1, H-CDR2 and H-CDR3 of a monoclonal antibody produced by aDHIC2 4-A10 hybridoma having ATCC accession number PTA-XXXX; and a lightchain comprising an L-CDR-1, L-CDR2 and L-CDR3 of a monoclonal antibodyproduced by the HIC0 4-F9, hybridoma having ATCC accession numberPTA-XXXX, wherein the monoclonal antibody specifically binds pancreaticductal cells.
 2. The isolated monoclonal antibody of claim 1, whereinthe monoclonal antibody is produced by the DHIC2 4-A10 hybridoma havingATCC accession number PTA-XXXX.
 3. The isolated monoclonal antibody ofclaim 1, the antibody comprising a human framework region.
 4. Anisolated antigen-binding fragment of the isolated monoclonal antibody ofclaim 1, wherein the antigen binding fragment specifically bindspancreatic ductal cells.
 5. The isolated antigen-binding fragment of themonoclonal antibody of claim 4, wherein the antigen-binding fragment isa scFV, a Fv, a Fab, a F(ab′)₂ or a scFV₂ fragment.
 6. The isolatedmonoclonal antibody of claim 1, or an antigen-binding fragment thereof,conjugated to an effector molecule.
 7. The isolated monoclonal antibodyor antigen-binding fragment of claim 6, wherein the effector molecule isa detectable marker or a toxin.
 8. The isolated monoclonal antibody orantigen-binding fragment of claim 7, wherein the detectable marker is afluorescent marker or a radiolabel.
 9. The isolated antibody orantigen-binding fragment of claim 7, wherein the toxin comprises ricinA, abrin, diphtheria toxin or a subunit thereof, Pseudomonas exotoxin ora portion thereof, saporin, restrictocin or gelonin.
 10. An isolatednucleic acid encoding the monoclonal antibody of claim 1, or encoding anantigen binding fragment of the monoclonal antibody.
 11. The isolatednucleic acid of claim 10, operably linked to a promoter.
 12. Anexpression vector comprising the nucleic acid of claim
 10. 13. Anisolated host cell transformed with the expression vector of claim 12.14. A hybridoma producing the monoclonal antibody of claim
 1. 15. Acomposition comprising the monoclonal antibody of claim 1 or anantigen-binding fragment thereof and physiologically acceptable carrier.16. A method for detecting a pancreatic ductal cell, the methodcomprising: contacting a cell of interest with the monoclonal antibodyof claim 1 or an antigen binding fragment thereof; and detecting bindingof the monoclonal antibody or the antigen binding fragment thereof tothe cell, wherein binding of the monoclonal antibody or the antigenbinding fragment thereof to the cell indicates that the cell is apancreatic ductal cell.
 17. The method of claim 16, wherein thepancreatic ductal cell is in vitro.
 18. The method of claim 16, whereinthe pancreatic ductal cell is in vivo.
 19. The method of claim 17,wherein the pancreatic ductal cell is isolated from a subject that has apancreatic endocrine cell tumor or diabetes.
 20. A method of isolating apancreatic ductal cell, comprising: contacting a suspension ofpancreatic cells with the monoclonal antibody of claim 1 or an antigenbinding fragment thereof to form an immune complex; and isolating theimmune complex, thereby isolating the pancreatic ductal cell.
 21. Themethod of claim 20, further comprising contacting the immune complexwith a second antibody that specifically binds the monoclonal antibodyof claim 3 or the antigen-binding fragment thereof.
 22. The method ofclaim 20, wherein the monoclonal antibody of claim 1 or theantigen-binding fragment thereof is coupled to a detectable marker. 23.The method of claim 21, wherein the second antibody is coupled to adetectable marker.
 24. The method of claim 22, wherein the detectablemarker is a fluorescent marker.
 25. A method for isolating a pancreaticductal cell, the method comprising: contacting cells with the antibodyof claim 1; detecting binding of the antibody to the cells; andisolating a cell bound to the antibody, thereby isolating the pancreaticductal cell.
 26. The method of claim 16, wherein the cell is from ahuman cadaveric islet.
 27. The method of claim 16, wherein the cell isdifferentiated from an embryonic stem cell, an adult stem cell or aprogenitor cell.
 28. The method of claim 20, wherein the cells are froma human cadaveric islet.
 29. The method of claim 20, wherein the cellsare differentiated from an embryonic stem cells, an adult stem cell or aprogenitor cell.